Compositions and methods for the identification, assessment, prevention and therapy of cancer

ABSTRACT

The subject matter relates to newly discovered nucleic acid molecules and proteins associated with thymic lymphoma and hamartomatous tumors. Compositions and methods for detecting, characterizing, preventing, and treating human thymic lymphoma and hamartomatous tumors are provided.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Application No. 60/897,088 filed on 24 Jan. 2007, which application is incorporated herein in its entirety by this reference.

BACKGROUND

The phosphoinositide 3-kinase (PI3K)-AKT pathway is activated in human cancers, commonly through genetic alterations of its many signalling components. The p110α catalytic subunit of PI3K is mutated or amplified and overexpressed in ovarian, breast, cervical, brain and colorectal cancers (Campbell et al., 2004; Samuels et al., 2004). AKT is amplified and overexpressed in breast, ovarian and pancreatic cancer (Bellacosa et al., 1995; Cheng et al., 1992; Miwa et al., 1996; Ruggeri et al., 1998). PTEN, the key PI3K-AKT pathway antagonist, is frequently targeted for somatic inactivation in a broad spectrum of human cancers (Cully et al., 2006). Moreover, germline PTEN mutations define three related syndromes (Cowden disease, Lhermitte-Duclos disease, and Bannayan-Zonana syndrome) characterized by hamartomatous growths of multiple cell types, increased cancer incidence and developmental defects (Wanner et al., 2001). PTEN's diverse tumor suppressor role in different cell lineages is further evidenced by the cancer-prone condition in germline and conditional Pten mutant mice, which develop a broad range of tumors including carcinomas of the skin, prostate, colon, breast, and endometrium, as well as thymic lymphomas, among other cancers (Di Cristofano et al., 1998; Li et al., 2002; Podsypanina et al., 1999; Suzuki et al., 2001; Wang et al., 2003; You et al., 2002).

In addition to a direct role in tumor development, the PI3K-AKT signalling network has been linked to endothelial cell homeostasis (Shiojima and Walsh, 2002). At least one of the three AKT homologues is highly expressed in endothelial cells and stimulates eNOS and survivin expression to promote survival (Dimmeler et al., 1999; Fulton et al., 1999; Tran et al., 2002). Consistent with these observations, one of the PTEN germline mutation syndromes, Bannayan-Zonana, is characterized by hemangiomas (hamartomas of the endothelial cell lineage) in diverse tissues including the deep viscera (Wanner et al., 2001).

The wide range of benign to malignant phenotypes mediated by PI3K-PTEN-AKT signalling is consistent with the existence of diverse downstream effectors that are likely to be differentially utilized in conferring neoplastic phenotypes in distinct cell lineages. Among such potential effectors are AKT phosphorylation targets TSC2, GSK3 and the FoxOs (Brunet et al., 1999; Cross et al., 1995; Inoki et al., 2002). Recently, much attention has been focused on TSC2 and the mTOR signalling axis as mounting pharmacological evidence suggests that this axis may be the prime effector of the PI3K-AKT pathway, with TSC2 serving as a central node linking LKB1-AMPK and PI3K-AKT with mTOR (Hay, 2005). Indeed, the potent anti-neoplastic impact of pharmacologic mTOR inhibition has raised questions as to the relevance of other downstream AKT targets, particularly the FoxOs, in the development of cancer. On the other hand, the FoxO effector arm has been shown to control cell number in Drosophila (Junger et al., 2003; Puig et al., 2003), and recent studies have implicated the FoxO transcription factors in mediating some of the growth and survival-promoting effects of AKT signaling in endothelial cells (Potente et al., 2005; Skurk et al., 2004). Therefore, the relevance of FoxO transcription factors in cancer and their roles in normal tissue homeostasis remain to be elucidated.

Whereas C. elegans and D. melanogaster contain a single FoxO gene (DAF-16 and dFOXO, respectively), mice and humans possess three highly related FoxO homologues (FoxO1, FoxO3, and FoxO4) with overlapping patterns of expression and transcriptional activities (Anderson et al., 1998; Biggs et al., 2001; Furuyama et al., 2000). A fourth more distantly related mammalian FoxO family member, FoxO6, has been identified, although it appears to be regulated by distinct mechanisms and its expression is more highly restricted to the brain (Jacobs et al., 2003; van der Heide et al., 2005). Activation of PI3K by extracellular growth signals leads to FoxO phosphorylation by Akt, whereupon the FoxOs are translocated from the nucleus to the cytoplasm and thereby inactivated.

In the nucleus, the FoxOs regulate cell survival and cell cycle progression through direct positive and negative transcriptional control of specific gene targets that are wired into diverse cancer regulatory pathways. The FoxOs promote apoptosis via up-regulation of FasL (Alvarez et al., 2001; Brunet et al., 1999; Siegel et al., 2000) and Bim (Dijkers et al., 2002; Dijkers et al., 2000a; Stahl et al., 2002), as well as down-regulation of the pro-survival factor BCL-xL (Tang et al., 2002). In cell cycle regulation, enforced FoxO expression results in G₁ arrest through increased expression of the cyclin-dependent kinase inhibitor p27^(kip1) and down-regulation of D-type cyclins (Medema et al., 2000) (Schmidt et al., 2002). The FoxOs have also been linked to other cancer-relevant pathways, such as the NFκB and TGF-β pathways. The IκB kinase phosphorylates and inhibits the FoxO factors, rationalizing some of the growth-promoting properties of IκB kinase (Hu et al., 2004). The FoxO factors also associate with Smad proteins, which are activated by TGF-β signaling (Seoane et al., 2004), to enhance transcription of the cell cycle inhibitor, p21^(Cip1).

The FoxO1, FoxO3, and FoxO4 proteins behave similarly in biochemical studies, regulate common target genes, and bind to the same target DNA sequence (Biggs et al., 2001; Brunet et al., 1999; Furuyama et al., 2000). At the same time, mouse FoxO knockout mutants have revealed unique roles for the FoxOs, such as the requirement for FoxO3 in the regulation of ovarian primordial follicle activation (Castrillon et al., 2003; Hosaka et al., 2004) and for FoxO1 in vascular development during embryogenesis (Furuyama et al., 2004; Potente et al., 2005). However, while the three FoxOs serve some discrete functions, they are also likely to have significant functional redundancies, particularly as they are broadly expressed during embryonic development and in adult tissues (Furuyama et al., 2000). In this regard, conventional genetic analysis can fail to uncover important biological functions among closely-related gene families, as has been demonstrated for the Rb/p107/p130 (Lee et al., 1996; Sage et al., 2000) and p53/p63/p73 gene families (Flores et al., 2005; Flores et al., 2002). Indeed, many analogies can be drawn between the FoxO and the Rb, and p53 gene families, all of which regulate cell survival and growth pathways relevant to cancer and consist of members with common physiological roles.

In the context of the above biochemical and biological data linking the FoxOs to key cancer signaling pathways, initial indications that various FoxO knockout mice do not show an overt tumor-prone phenotype were somewhat unanticipated (Castrillon et al., 2003; Furukawa-Hibi et al., 2002; Hosaka et al., 2004; Lin et al., 2004). While this may relate to the physical and functional relatedness and overlapping patterns of expression of the FoxO members, it is formally possible that the FoxO arm of the PI3K-AKT signaling network plays a relatively minor role in cancer suppression and vascular biology relative to other AKT downstream targets. To address these issues, we have generated conditional alleles for all three FoxO members with which to conduct a systematic evaluation of FoxO family function in vivo.

SUMMARY

The invention relates to cancer markers (hereinafter “markers” or “markers of the inventions”), which are listed in Tables 1, 2, 3 and 4. These markers were identified as targets of the FoxO transcription factors through a systematic evaluation of FoxO family function performed in vivo. The invention provides nucleic acids and proteins that are encoded by or correspond to the markers (hereinafter “marker nucleic acids” and “marker proteins,” respectively). Tables 1, 2, 3 and 4 provide the sequence identifiers of the sequences of such marker nucleic acids and proteins listed in the accompanying Sequence Listing. The invention further provides antibodies, antibody derivatives and antibody fragments which bind specifically with such proteins and/or fragments of the proteins.

The invention also relates to various methods, reagents and kits for diagnosing, staging, prognosing, monitoring and treating thymic lymphoma. “Thymic lymphoma” as used herein includes but is not necessarily limited to Precursor T-lymphoblastic lymphoma, Lymphoblastic neoplasms of T-cell origin, Hodgkin lymphoma (e.g. Nodular lymphocyte-predominant Hodgkin lymphoma, Classical Hodgkin lymphoma), Histiocytic lymphomas, Carcinoid tumors, Germ cell tumors (e.g. seminoma, teratoma, embryonal cell carcinoma, choriocarcinoma), Primary mediastinal large B-cell lymphoma, and all other types of cancers, malignancies, and transformations associated with thymocytic cells. In one embodiment, the invention provides a diagnostic method of assessing whether a patient has thymic lymphoma, or has higher than normal risk for developing thymic lymphoma, comprising the steps of comparing the level of expression of a marker of the invention in a patient sample and the normal level of expression of the marker in a control, e.g., a sample from a patient without thymic lymphoma. A significantly higher level of expression of any marker listed in Table 1, or a significantly lower level of expression any marker listed in Table 2, in the patient sample as compared to the normal level is an indication that the patient is afflicted with thymic lymphoma, or has higher than normal risk for developing thymic lymphoma.

The invention further relates to various methods, reagents and kits for diagnosing, staging, prognosing, monitoring and treating hamartomatous tumors. “Hamartomatous tumors” as used herein includes but is not necessarily limited to benign tumors arising in a vascular organ (e.g. lung, liver, uterus, hypothalmus, kidney, spleen, skeletal muscle, abdominal wall, adrenal gland, bone marrow, omentum, lymph node, skin), malignant angiosarcomas derived from such benign tumors, hemangiomas, and hamartomas resulting from Cowden syndrome, Lhermitte-Duclos disease, and Bannayan-Zonana syndrome. In one embodiment, the invention provides a diagnostic method of assessing whether a patient has hamartomatous tumors, or has higher than normal risk for developing hamartomatous tumors, comprising the steps of comparing the level of expression of a marker of the invention in a patient sample and the normal level of expression of the marker in a control, e.g., a sample from a patient without hamartomatous tumors. A significantly higher level of expression of any marker listed in Table 3, or a significantly lower level of expression any marker listed in Table 4, in the patient sample as compared to the normal level is an indication that the patient is afflicted with hamartomatous tumors, or has higher than normal risk for developing hamartomatous tumors.

As used herein, “thymocytic cell” refers both to T lymphocytes and their precursors (i.e. any cell that is, or is destined to become a T lymphocyte), and includes but is not limited to immature thymocytes (e.g. lacking expression of CD4 and CD8 molecules), intermediate-stage thymocytes (e.g. expressing both CD4 and CD8 molecules), mature CD4-positive cells, and mature CD8-positive cells.

As used herein, “thymocytic cell-associated tissue” refers to any tissue that contains or is comprised of thymocytic cells, in part or in full.

As used herein, “subject” refers to any healthy animal, mammal or human, or any animal, mammal or human afflicted with thymic lymphoma and/or hamartomatous tumors. The term “subject” is interchangeable with “patient”.

According to the invention, the markers in Tables 1 and 2 are selected such that the positive predictive value of the methods of the invention is at least about 10%, preferably about 25%, more preferably about 50% and most preferably about 90%. Also acceptable for use in the methods of the invention are markers that are differentially expressed, as compared to normal cells, by at least two-fold in at least about 20%, more preferably about 50% and most preferably about 75% of any of the following conditions: Precursor T-lymphoblastic lymphoma, Lymphoblastic neoplasms of T-cell origin, Hodgkin lymphoma (e.g. Nodular lymphocyte-predominant Hodgkin lymphoma, Classical Hodgkin lymphoma), Histiocytic lymphomas, Carcinoid tumors, Germ cell tumors (e.g. seminoma, teratoma, embryonal cell carcinoma, choriocarcinoma), Primary mediastinal large B-cell lymphoma, and all other types of cancers, malignancies, and transformations associates with the thymocytic cell line.

According to the invention, the markers in Tables 3 and 4 are selected such that the positive predictive value of the methods of the invention is at least about 10%, preferably about 25%, more preferably about 50% and most preferably about 90%. Also acceptable for use in the methods of the invention are markers that are differentially expressed, as compared to normal cells, by at least two-fold in at least about 20%, more preferably about 50% and most preferably about 75% of any of the following conditions: Hamartomatous tumors including benign tumors arising in a vascular organ (e.g. lung, liver, uterus, hypothalmus, kidney, spleen, skeletal muscle, abdominal wall, adrenal gland, bone marrow, omentum, lymph node, skin), malignant angiosarcomas derived from such benign tumors, and hamartomas resulting from Cowden syndrome, Lhermitte-Duclos disease, and Bannayan-Zonana syndrome.

In one diagnostic method of assessing whether a patient is afflicted with thymic lymphoma or hamartomatous tumors (e.g., new detection (“screening”), detection of recurrence, reflex testing), the method comprises comparing:

a) the level of expression of a marker of the invention in a patient sample, and

b) the normal level of expression of the marker in a control sample lacking lymphoma or hamartomatous tumors.

A significantly higher level of expression of a marker listed in Table 1, or a significantly lower level of expression of a marker listed in Table 2, in the patient sample as compared to the normal level is an indication that the patient is afflicted with thymic lymphoma.

A significantly higher level of expression of a marker listed in Table 3, or a significantly lower level of expression of a marker listed in Table 4, in the patient sample as compared to the normal level is an indication that the patient is afflicted with hamartomatous tumors.

The invention also provides diagnostic methods for assessing the efficacy of a therapy for inhibiting thymic lymphoma or the development of hamartomatous tumors in a patient. Such methods comprise comparing:

a) expression of a marker of the invention in a first sample obtained from the patient prior to providing at least a portion of the therapy to the patient, and

b) expression of the marker in a second sample obtained from the patient following provision of the portion of the therapy.

For the markers presented in Tables 1 and 3, a significantly lower level of expression of the marker in the second sample relative to that in the first sample is an indication that the therapy is efficacious for inhibiting thymic lymphoma or hamartomatous tumors in the patient, respectively.

For the markers presented in Tables 2 and 4, a significantly higher level of expression of the marker in the second sample relative to that in the first sample is an indication that the therapy is efficacious for inhibiting thymic lymphoma or hamartomatous tumors in the patient, respectively.

It will be appreciated that in these methods the “therapy” may be any therapy for treating lymphoma or hamartomatous tumors including, but not limited to, chemotherapy, radiation therapy, surgical removal of tumor tissue, gene therapy and biologic therapy such as the administering of antibodies and chemokines. Thus, the methods of the invention may be used to evaluate a patient before, during and after therapy, for example, to evaluate the reduction in tumor burden.

In a particular embodiment, the diagnostic methods are directed to therapy using a chemical or biologic agent. These methods comprise comparing:

a) expression of a marker of the invention in a first sample obtained from the patient and maintained in the presence of the chemical or biologic agent, and

b) expression of the marker in a second sample obtained from the patient and maintained in the absence of the agent.

For the markers presented in Tables 1 and 3, a significantly lower level of expression of the marker in the second sample relative to that in the first sample is an indication that the agent is efficacious for inhibiting thymic lymphoma or hamartomatous tumors in the patient, respectively. In one embodiment, the first and second samples can be portions of a single sample obtained from the patient or portions of pooled samples obtained from the patient.

For the markers presented in Tables 2 and 4, a significantly higher level of expression of the marker in the second sample relative to that in the first sample is an indication that the agent is efficacious for inhibiting thymic lymphoma or hamartomatous tumors in the patient, respectively. In one embodiment, the first and second samples can be portions of a single sample obtained from the patient or portions of pooled samples obtained from the patient.

The invention additionally provides a monitoring method for assessing the progression of thymic lymphoma, or the development of hamartomatous tumors, in a patient, the method comprising:

a) detecting in a patient sample at a first time point, the expression of a marker of the invention;

b) repeating step a) at a subsequent time point in time; and

c) comparing the level of expression detected in steps a) and b), and therefrom monitoring the progression of thymic lymphoma or development of hamartomatous tumors in the patient.

For the markers presented in Table 1, a significantly higher level of expression of the marker in the sample at the subsequent time point from that of the sample at the first time point is an indication that the thymic lymphoma has progressed, whereas a significantly lower level of expression is an indication that the thymic lymphoma has regressed.

For the markers presented in Table 2, a significantly lower level of expression of the marker in the sample at the subsequent time point from that of the sample at the first time point is an indication that the thymic lymphoma has progressed, whereas a significantly higher level of expression is an indication that the thymic lymphoma has regressed.

For the markers presented in Table 3, a significantly higher level of expression of the marker in the sample at the subsequent time point from that of the sample at the first time point is an indication that the development of hamartomatous tumors has progressed, whereas a significantly lower level of expression is an indication that the development of hamartomatous tumors has regressed.

For the markers presented in Table 4, a significantly lower level of expression of the marker in the sample at the subsequent time point from that of the sample at the first time point is an indication that the development of hamartomatous tumors has progressed, whereas a significantly higher level of expression is an indication that the development of hamartomatous tumors has regressed.

The invention further provides a diagnostic method for determining whether a thymic lymphoma or hamartomatous tumor has metastasized or is likely to metastasize in the future, the method comprising comparing:

a) the level of expression of a marker of the invention in a patient sample, and

b) the normal level (or non-metastatic level) of expression of the marker in a control sample.

For the markers presented in Tables 1 and 3, a significantly higher level of expression in the patient sample as compared to the normal level (or non-metastatic level) is an indication that the thymic lymphoma or hamartomatous tumor, respectively, has metastasized or is likely to metastasize in the future.

For the markers presented in Tables 2 and 4, a significantly lower level of expression in the patient sample as compared to the normal level (or non-metastatic level) is an indication that the thymic lymphoma or hamartomatous tumor, respectively, has metastasized or is likely to metastasize in the future.

The invention moreover provides a test method for selecting a composition for inhibiting thymic lymphomas or the development of hamartomatous tumors in a patient. This method comprises the steps of:

a) obtaining a sample comprising cancer cells from the patient;

b) separately maintaining aliquots of the sample in the presence of a plurality of test compositions;

c) comparing expression of a marker of the invention in each of the aliquots; and

d) for the markers presented in Tables 1 and 3, selecting one of the test compositions which significantly reduces the level of expression of the marker in the aliquot containing that test composition, relative to the levels of expression of the marker in the presence of the other test compositions.

e) for the markers presented in Tables 2 and 4, selecting one of the test compositions which significantly increases the level of expression of the marker in the aliquot containing that test composition, relative to the levels of expression of the marker in the presence of the other test compositions.

The invention additionally provides a test method of assessing the carcinogenic potential of a compound. This method comprises the steps of:

a) maintaining separate aliquots of cells in the presence and absence of the compound; and

b) comparing expression of a marker of the invention in each of the aliquots.

For the markers presented in Tables 1 and 3, a significantly higher level of expression of the marker in the aliquot maintained in the presence of the compound, relative to that of the aliquot maintained in the absence of the compound, is an indication that the compound possesses carcinogenic potential.

For the markers presented in Tables 2 and 4, a significantly lower level of expression of the marker in the aliquot maintained in the presence of the compound, relative to that of the aliquot maintained in the absence of the compound, is an indication that the compound possesses carcinogenic potential.

In addition, the invention further provides a method of inhibiting thymic lymphoma or hamartomatous tumors in a patient. This method comprises the steps of:

a) obtaining a sample comprising cancer cells from the patient;

b) separately maintaining aliquots of the sample in the presence of a plurality of compositions;

c) comparing expression of a marker of the invention in each of the aliquots; and

d) for the markers presented in Tables 1 and 3, administering to the patient at least one of the compositions which significantly lowers the level of expression of the marker in the aliquot containing that composition, relative to the levels of expression of the marker in the presence of the other compositions.

e) for the markers presented in Tables 2 and 4, administering to the patient at least one of the compositions which significantly elevates the level of expression of the marker in the aliquot containing that composition, relative to the levels of expression of the marker in the presence of the other compositions.

In the aforementioned methods, the samples or patient samples comprise cells obtained from the patient.

According to the invention, the level of expression of a marker of the invention in a sample can be assessed, for example, by detecting the presence in the sample of:

the corresponding marker protein (e.g., a protein having one of the sequences set forth as “SEQ ID NO (AAs)”, or a fragment of the protein (e.g. by using a reagent, such as an antibody, an antibody derivative, an antibody fragment or single-chain antibody, which binds specifically with the protein or protein fragment)

the corresponding marker nucleic acid (e.g. a nucleotide transcript having one of the nucleic acid sequences set forth as “SEQ ID NO (nts)”, or a complement thereof), or a fragment of the nucleic acid (e.g. by contacting transcribed polynucleotides obtained from the sample with a substrate having affixed thereto one or more nucleic acids having the entire or a segment of the nucleic acid sequence of any of the SEQ ID NO (nts), or a complement thereof)

a metabolite which is produced directly (i.e., catalyzed) or indirectly by the corresponding marker protein.

According to the invention, any of the aforementioned methods may be performed using a plurality (e.g. 2, 3, 5, or 10 or more) of markers for thymic lymphoma or hamartomatous tumors, including markers known in the art. In such methods, the level of expression in the sample of each of a plurality of markers, at least one of which is a marker of the invention, is compared with the normal level of expression of each of the plurality of markers in samples of the same type obtained from control humans not afflicted with thymic lymphoma or hamartomatous tumors. A significantly altered (i.e., increased or decreased as specified in the above-described methods using a single marker) level of expression in the sample of one or more markers of the invention, or some combination thereof, relative to that marker's corresponding normal or control level, is an indication that the patient is afflicted with thymic lymphoma or hamartomatous tumors. For all of the aforementioned methods, the marker(s) may be selected such that the positive predictive value of the method is at least about 10%.

In a further aspect, the invention provides an antibody, an antibody derivative, or an antibody fragment, which binds specifically with a marker protein or a fragment of the protein. The invention also provides methods for making such antibody, antibody derivative, and antibody fragment. Such methods may comprise immunizing a mammal with a protein or peptide comprising the entirety, or a segment of 10 or more amino acids, of a marker protein, wherein the protein or peptide may be obtained from a cell or by chemical synthesis. The methods of the invention also encompass producing monoclonal and single-chain antibodies, which would further comprise isolating splenocytes from the immunized mammal, fusing the isolated splenocytes with an immortalized cell line to form hybridomas, and screening individual hybridomas for those that produce an antibody that binds specifically with a marker protein or a fragment of the protein.

In another aspect, the invention relates to various diagnostic and test kits. In one embodiment, the invention provides a kit for assessing whether a patient is afflicted with thymic lymphoma or hamartomatous tumors. The kit comprises a reagent for assessing expression of a marker of the invention. In another embodiment, the invention provides a kit for assessing the suitability of a chemical or biologic agent for inhibiting thymic lymphoma or hamartomatous tumors in a patient. Such a kit comprises a reagent for assessing expression of a marker of the invention, and may also comprise one or more of such agents. In a further embodiment, the invention provides kits for assessing the presence of thymic lymphoma or hamartomatous tumor cells or treating thymic lymphoma or hamartomatous tumors. Such kits comprise an antibody, an antibody derivative, or an antibody fragment, which binds specifically with a marker protein, or a fragment of the protein. Such kits may also comprise a plurality of antibodies, antibody derivatives, or antibody fragments wherein the plurality of such antibody agents binds specifically with a marker protein, or a fragment of the protein.

In an additional embodiment, the invention also provides a kit for assessing the presence of thymic lymphoma or hamartomatous tumor cells, wherein the kit comprises a nucleic acid probe that binds specifically with a marker nucleic acid or a fragment of the nucleic acid. The kit may also comprise a plurality of probes, wherein each of the probes binds specifically with a marker nucleic acid, or a fragment of the nucleic acid.

In a further aspect, the invention relates to methods for treating a patient afflicted with thymic lymphoma or hamartomatous tumors or at risk of developing thymic lymphoma or hamartomatous tumor. Such methods may comprise reducing the expression and/or interfering with the biological function of a marker of the invention. In one embodiment, the method comprises providing to the patient an antisense oligonucleotide or polynucleotide complementary to a marker nucleic acid, or a segment thereof. For example, an antisense polynucleotide may be provided to the patient through the delivery of a vector that expresses an anti-sense polynucleotide of a marker nucleic acid or a fragment thereof. In another embodiment, the method comprises providing to the patient an antibody, an antibody derivative, or antibody fragment, which binds specifically with a marker protein or a fragment of the protein.

It will be appreciated that the methods and kits of the present invention may also include known cancer markers including known cancer markers for thymic lymphoma or hamartomatous tumors. It will further be appreciated that the methods and kits may be used to identify cancers other than thymic lymphoma or hamartomatous tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a-f depict thymic lymphomas in mice following somatic deletion of all three FoxO genes. a, Thymic lymphoma-free survival of pI-pC treated Mx-Cre⁺ mice (Red line, n=22) and controls (black line, n=72) representing combined pI-pC treated control genotypes including: Mx-Cre⁺;FoxO1^(L/L) (n=11), Mx-Cre⁺;FoxO1/3^(L/L) (n=14), Mx-Cre⁺;FoxO1/4^(L/L) (n=11), and all Mx-Cre⁻ controls (n=36, littermates of the other genotypes). No lymphomas were observed in these controls up to 100 weeks of age. b, Histology and tissue infiltration of representative thymic lymphoma in Mx-Cre⁺ mouse, hematoxylin and eosin stains. (i) Primary thymic lymphoma and infiltration in (ii) liver, (iii) lung, and (iv) bone marrow. Scale bars: 200 μm (i and ii) and 100 μm (iii and iv). c, FoxO1, FoxO3, and FoxO4 gene deletions in thymic lymphomas by PCR analysis. Lanes: 1) Mx-Cre⁻ normal thymus, 2) Mx-Cre⁺ normal thymus, and 3) Mx-Cre⁺ thymic lymphoma. d, Reduction of mRNA levels of FoxO1, FoxO3, and FoxO4 in Mx-Cre⁺ endothelial cells. Quantitative real-time PCR analysis was performed on Mx-Cre⁻ thymi (n=2, 7 weeks), Mx-Cre⁺ thymi (n=2, 7 weeks), and Mx-Cre⁺ thymic lymphomas (n=2, 19 and 30 weeks post pI-pC); the relative reduction of FoxO levels in relation to Mx-Cre⁻ thymi is shown. e, Western blot analysis performed on lysates from representative Mx-Cre⁻ and Mx-Cre⁺ thymi and thymic lymphoma samples. f, Flow cytometric analysis of representative thymic lymphoma. Cells were assayed for CD4, CD8, CD3, CD44, CD25, CD19, Gr-1, and Mac-1 expression.

FIGS. 2a-d depict systemic hemangiomas in mice following somatic deletion of all three FoxO alleles. a, Overall survival of Mx-Cre⁺ mice, other single- or double-floxed genotype combinations, and Mx-Cre⁻ littermates controls. Blue squares indicate deaths due to thymic lymphomas. P value indicates comparison between Mx-Cre⁺ mice (red) and all Mx-Cre⁻ controls (black). b, Age-dependent progression of uterine vascular lesions in Mx-Cre⁺ females. Panels (from left to right): Mx-Cre⁻ (negative control) at 30 weeks of age; Mx-Cre⁺ at 30 weeks; Mx-Cre⁺ at 43 weeks. Scale Bar=5 mm. c, Histologic examination of systemic vascular lesions, hematoxylin and eosin stains. Genotypes are Mx-Cre⁺ unless otherwise noted. i, uterus, low magnification. Asterisk indicates the uterine lumen; ii, uterine hemangioma (inset shows histologically-benign endothelium of hemangioma vascular channels; arrow indicates endothelial cell); iii, abdominal muscle hemangioma; iv, liver hemangioma from Mx-Cre⁺ mouse; v-vi, angiosarcoma from Mx-Cre⁺ mouse. Scale bars: 500 μm (i and v), 400 μm (iv), 200 μm (iii), and 50 μm (ii and vi). d, Fluorescence micrographs showing the vasculature of abdominal muscle (i-iv), uterine horn (v-viii), and lung (ix-x) after vascular perfusion of fluorescein-labeled lectin (blue: DAPI labelled nuclei) in Mx-Cre⁻ (left) and Mx-Cre⁺ (right) mice. Note increased endothelial cell density as early as 7 weeks in muscle and uterine horn (ii, vi) but lack of changes in lung as late as 31 weeks. Scale bar=50 μm.

FIGS. 3a-b depict functional studies of thymocytes and endothelial cells rendered null for all three FoxOs. a, Increased proliferation and defective induction of apoptosis in Mx-Cre⁺ thymocytes. Thymocytes from 8-week old mice (n=3, each genotype) were plated and the indicated mitogens or apoptotic stimuli were applied. Experiment was performed twice with similar results; data from a representative experiment is shown. Survival % was calculated after 18 hrs of treatment (5 Gy: γ-irradiation; Dex: 1 μM Dexamethasone). b, Analysis of viability of endothelial cells. MTT assays were performed on Mx-Cre⁻ and Mx-Cre⁺ lung (n=2) and liver (n=3) ECs. Y axis represents normalized response (%) based on the spectophotometric signal at OD595. Results represent two independent experiments. For a and b, data are mean±SEM. White: Mx-Cre⁻; black: Mx-Cre⁺.

FIGS. 4a-d depict the regulation of Sprouty2 expression in liver ECs by FoxOs. a, Multiple conserved FoxO binding sites (predicted) in vicinity of Sprouty2 gene; ˜11 kb of the genomic region is shown. Five of these ten sites were conserved in at least three species including human and mouse. 8 kb upstream+2 kb downstream of gene surrounding the transcriptional start site and the 5 kb region downstream of the transcriptional end site were scanned for FoxO consensus binding sequences by a position weight-matrix approach (see methods). Sites were scored by 10-based logarithm of the likelihood ratio between the FoxO and the background model. b. The FoxO BE occupancy by FoxOs. Soluble chromatin was prepared from Mx-Cre⁻ and Mx-Cre⁺ liver ECs and immunoprecipitated with mixture of antibodies against all three FoxOs. Anti-RNA polymerase II (pol) and anti-FoxO immunoprecipitated DNA (FoxO) was amplified using primer pairs giving rise to ˜110 bp products (a-d, illustrated in panel a). c, Quantitative real-time PCR analysis for Sprouty2. Expression of Sprouty2 is tightly correlated with FoxO deletion (Mx-Cre⁺) in liver ECs but not in lung ECs (**, p<0.01); error bars=standard error. d. Immunoblot analysis for Sprouty2. Expression of Sprouty2 is lower in FoxO deletion (Mx-Cre⁺) in liver ECs but not in lung ECs compared with control ECs. Duplicate loading was shown for each EC line. Results shown represent multiple experiments.

FIGS. 5a-e depict the regulation by FoxOs of the liver EC angiogenic response through Sprouty2. a, Growth advantage of Mx-Cre⁺ (right upper) over Mx-Cre⁻ liver ECs (left upper) is evident after 10 days of culture. Sprouty2 knockdown in Mx-Cre⁻ liver ECs rendered growth advantage (right lower, shSpry2). Bar=15 μm. b, TUNEL and BrdU assays showing decreased apoptosis and increased proliferation in Mx-Cre⁺ liver ECs. Knockdown of Sprouty2 (shSpry2_1 &_2, shRNAs) in Mx-Cre⁻ liver ECs phenocopies both the cell growth and apoptosis phenotypes seen in Mx-Cre⁺ liver ECs; v=vector only control (*, p<0.01; **, p<0.05); error bars represent ±s.e.c, knock-down of endogenous Sprouty2 protein expression in Mx-Cre⁻ liver ECs by shSpry2. Duplicate loadings of each treatment are shown. Note that knockdown level of Sprouty2 is comparable to that of Mx-Cre⁺ liver ECs. Results represent multiple independent experiments. d, Correlation of Sprouty2 expression with cyclinD1, p21, p15, Bim level in liver ECs by quantitative PCR analysis. Note that Mx-Cre⁺ liver ECs have decreased Sprouty2 expression associated with higher cyclinD1, and lower p21, p15 and Bim expression (upper panels). Knockdown of Sprouty2 with two different shRNAs (shSpry2_1 &_2) closely recapitulates the magnitude of the above differences for cyclinD1, p21, p15, and Bim relative to parental untreated (p) and vector-only (v) controls (lower panels). Results shown are the average fold differences over controls in triplicate experiments; error bars represent ±s.e. e, VEGF-induced tube formation was more robust in Mx-Cre⁺ liver ECs (lower left) relative to Mx-Cre⁻ liver ECs (upper left). Sprouty2 knockdown enhanced response to VEGF in Mx-Cre⁻ liver ECs (upper right panel). Bar=100 μm. Average tubule length/HPF (±sd) measured by ImageJ software in multiple microscopic fields was plotted (*, p<0.01 versus vector control).

FIGS. 6a-d depict the generation of FoxO1 conditional and null alleles. a, Schematic of FoxO1 targeting vector. LoxP sites were inserted into intronic sites flanking the second exon of FoxO1. PCR primers are indicated with arrows. b, Southern analysis of DNA from ES clones to demonstrate recombination. DNA was digested with HindIII+SspI (left panel) and SpeI (right panel). c, PCR genotyping of wild type, floxed, and null alleles using primers a+b+d. d, Confirmation of FoxO1 deletion and loss of expression in FoxO1 null MEFs by Northern analysis.

FIGS. 7a-d depict the generation of FoxO4 conditional and null alleles. a, Schematic of FoxO4 targeting vector. LoxP sites were inserted into intronic sites flanking the first exon of FoxO4. PCR primers are indicated with arrows. b, Southern analysis of DNA from ES clones to demonstrate recombination. DNA was digested with XhoI+NcoI. c, PCR genotyping of wild type, floxed, and null alleles using primers d+e+m. d, Confirmation of FoxO4 deletion and loss of expression in FoxO4^(−/−) skeletal muscle by Northern analysis.

FIGS. 8a-h depict an analysis of FoxO germline knockouts. a, Overall survival of FoxO1^(−/+) mice. b, Overall survival of FoxO4^(−/−) mice. c, Overall survival of FoxO3^(−/−) mice. d, Overall survival of FoxO3^(−/−) mice with dermatitis. For d-f, FoxO3^(−/−) mice are divided into two groups: those sacrificed due to dermatitis, and those without dermatitis-associated morbidity. e, Tumor-free survival of FoxO3^(−/−) males. f, Tumor-free survival of FoxO3^(−/−) females. g, Lung tumor load (total tumor volume) in FoxO4^(+/+) (n=8) and FoxO4−/− (n=15) mice. h, Histology of FoxO4^(−/−) lung tumors, hematoxylin and eosin stains. Scale bars: 500 μm (low magnification) and 100 μm (high power).

FIGS. 9a-f depict tumor-free survival of single and compound (germline) FoxO mutant mice. a, FoxO1. b, FoxO4. c, FoxO3. d, FoxO3 with exclusion of dermatitis-associated deaths. e, Pituitary adenomas in FoxO3 females. Left to right: normal pituitary (FoxO3^(+/+)), small pituitary adenoma (FoxO3^(+/+)), and large pituitary adenoma (FoxO3⁻). Black dashed lines demarcate pituitary gland; white dashed lines demarcate pituitary adenomas. f, Pituitary tumor histology, hematoxylin and eosin stain. Adenoma shows monomorphic cell population. Scale bar: 100 μm.

FIGS. 10a-d depict an analysis of DMBA-treated FoxO germline knockouts. a, Survival of DMBA-treated FoxO4^(−/−) mice. Pups were treated with DMBA at day 7. Mice underwent full autopsies and histopathologic analysis. b, Tumor development in DMBA-treated FoxO4^(−/−) mice. Total numbers of each genotype are indicated. For the incidence of angiosarcomas in the uterus, percentages were calculated from total number of females only. c, Survival of DMBA-treated FoxO3^(−/−) mice. d, Tumor development in DMBA-treated FoxO3^(−/−) mice.

FIGS. 11a-c depict an analysis of compound FoxO3/FoxO4 germline knockouts. a, Overall survival of FoxO4^(−/−); FoxO3^(−/−) and FoxO4^(−/−); FoxO3^(−/+) littermates and FoxO4^(+/+); FoxO3^(+/+) control mice. Survival of FoxO3^(−/−) mice is plotted for comparison. b, Tumor-free survival of compound FoxO4^(−/−); FoxO3^(−/−) mice. c, Tumor spectra of mice with compound germline FoxO mutations. FoxO4^(−/−); FoxO3^(−/−) mice (n=31) were analyzed in relation to FoxO4^(−/−); FoxO3^(−/+) (n=26) littermates and FoxO4^(+/+); FoxO3^(+/+) mice (n=28).

FIGS. 12a-d depict abnormal vascular lesions in Mx-Cre⁺ mice and intermediate controls. a-d, hematoxylin and eosin-stained tissue sections. a, Mx-Cre⁺ mice (20-43 weeks). i, adrenal gland (medulla replaced by hemorrhage, *=region of hemorrhage; scale bar=400 μm), ii, Hemangiomatous change in liver (scale bar=200 μm), iii, bone marrow (left: bone marrow from Mx-Cre⁻ mouse; right: bone marrow from Mx-Cre⁺ mouse; scale bar=100 μm), iv, skin (scale bar=200 μm), v, perirenal fat hemangioma, (scale bar=200 μm), vi, hemorrhagic lymph node (scale bar=500 μm). b, Uterine horn from 60-week Mx-Cre⁺;FoxO1^(L/L). i, low power (scale bar=2 mm), ii, high power of boxed region from (i) (scale bar=500 μm). c, Uterine horn and perirenal fat hemangioma from 60-week Mx-Cre⁺;FoxO1/4^(L/L) mouse. i, uterine horn, low power (scale bar=2 mm); ii, uterine horn, high power (scale bar=500 μm); iii, perirenal fat hemangioma (scale bar=200 μm). d, Mx-Cre⁺;FoxO1/3^(L/L) mouse (50 weeks). Depicted are: uterine horn (low (i) and high (ii) power), adipose tissue (iii), and skin (iv). Scale bars: 500 μm, (i), 50 μm (ii), 200 μm (iii and iv).

FIGS. 13a-b depict broad Cre-mediated recombination in endothelium and most epithelia following pI-pC treatment as a result of the Mx-Cre transgene. a, Rosa26-lacZ reporter mice 6 months after pI-pC treatment. a, Liver (inset=control liver). b, Thymus. c, Lung. Liver, thymus, and lung parenchymal cells show very high levels of recombination. d, Pancreas. e, Salivary gland (submandibular). Both pancreas and salivary gland showed high levels of recombination in ductal epithelium, and a low frequency of recombination in acinar cells. f, Mammary gland with essentially complete recombination. g, Skin. h, Small intestine. Skin and small intestine epithelial cells exhibited subtotal recombination; distinct clones of positive cells were observed. i, Renal

tubules. j, Brain. k, Abdominal muscle. Brain and skeletal muscle exhibited a very low frequency of recombination, except for endothelial cells where recombination frequencies approached 100%, as was the case in other tissues. The blue staining surrounding myofibers corresponds to capillary endothelium. 1, uterus with high frequency of recombination in endothelial cells and in some myometrial cells, particularly those of the outer layer (bottom edge of figure). Scale bar=120 μm (d, e); 60 μm (a, f, g, h, i, k); 40 μm (b, c, j, 1). b, Quantitative real-time PCR analysis of FoxO deletion in Mx-Cre⁺ mice. Tissues were harvested from 2-3 sets each of Mx-Cre⁺ and Mx-Cre⁻ mice and RNA was isolated and analyzed for FoxO1, FoxO3, or FoxO4 expression.

FIG. 14 depicts decreased expression of Sprouty2 protein in vascular bed prone to developing hemangiomas. Panels show vascular endothelium of Mx-Cre⁺ skeletal muscle tissue 3 weeks after pI-pC treatment; tissue sections were co-stained with an endothelial marker as a positive control (MadCam).

DETAILED DESCRIPTION

The invention relates to newly discovered cancer markers associated with the cancerous state of thymic lymphoma or hamartomatous tumor cells. It has been discovered that the higher than normal level of expression of any of the markers provided in Table 1 or combination of these markers correlates with the presence of thymic lymphoma including pre-malignant conditions such as dysplasia, in a patient. Similarly, it has been discovered that the lower than normal level of expression of any of the markers provided in Table 2 or combination of these markers correlates with the presence of thymic lymphoma including pre-malignant conditions such as dysplasia, in a patient. It has further been discovered that the higher than normal level of expression of any of the markers provided in Table 3 or combination of these markers correlates with the presence of hamartomatous tumors in a patient. Similarly, it has been discovered that the lower than normal level of expression of any of the markers provided in Table 4 or combination of these markers correlates with the presence of hamartomatous tumors in a patient. Methods are provided for detecting the presence of thymic lymphoma or hamartomatous tumors in a sample, the absence of thymic lymphoma or hamartomatous tumors in a sample, the stage of a thymic lymphoma or a hamartomatous tumor, and other characteristics of thymic lymphoma or hamartomatous tumors that are relevant to prevention, diagnosis, characterization, and therapy of thymic lymphoma or hamartomatous tumors in a patient. Methods of treating thymic lymphoma or hamartomatous tumors are also provided.

Table 1 lists the markers of the invention which are over-expressed in thymic lymphoma cells compared to normal (i.e., non-cancerous) cells.

Table 2 lists the markers of the invention which are under-expressed in thymic lymphoma cells compared to normal (i.e., non-cancerous) cells.

Table 3 lists the markers of the invention which are over-expressed in hamartomatous tumor cells compared to normal (i.e., non-cancerous) cells.

Table 4 lists the markers of the invention which are under-expressed in hamartomatous tumor cells compared to normal (i.e., non-cancerous) cells.

Definitions

As used herein, each of the following terms has the meaning associated with it in this section.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

A “marker” is a gene whose altered level of expression in a tissue or cell from its expression level in normal or healthy tissue or cell is associated with a disease state, such as cancer. A “marker nucleic acid” is a nucleic acid (e.g., mRNA, cDNA) encoded by or corresponding to a marker of the invention. Such marker nucleic acids include DNA (e.g., cDNA) comprising the entire or a partial sequence of any of the nucleic acid sequences set forth in the Sequence Listing or the complement of such a sequence. The marker nucleic acids also include RNA comprising the entire or a partial sequence of any of the nucleic acid sequences set forth in the Sequence Listing or the complement of such a sequence, wherein all thymidine residues are replaced with uridine residues. A “marker protein” is a protein encoded by or corresponding to a marker of the invention. A marker protein comprises the entire or a partial sequence of any of the sequences set forth in the Sequence Listing. The terms “protein” and “polypeptide” are used interchangeably.

The term “body fluid” refers to fluids that are excreted or secreted from the body as well as fluid that are normally not (e.g. amniotic fluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen and earwax, cowper's fluid or pre-ejaculatory fluid, chyle, chyme, stool, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, vomit).

The term “probe” refers to any molecule which is capable of selectively binding to a specifically intended target molecule, for example, a nucleotide transcript or protein encoded by or corresponding to a marker. Probes can be either synthesized by one skilled in the art, or derived from appropriate biological preparations. For purposes of detection of the target molecule, probes may be specifically designed to be labeled, as described herein. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

The “normal” level of expression of a marker is the level of expression of the marker in cells of a human subject or patient not afflicted with thymic lymphoma or hamartomatous tumors. An “over-expression” or “significantly higher level of expression” of a marker refers to an expression level in a test sample that is greater than the standard error of the assay employed to assess expression, and is preferably at least twice, and more preferably three, four, five or ten times the expression level of the marker in a control sample (e.g., sample from a healthy subjects not having the marker associated disease) and preferably, the average expression level of the marker in several control samples.

A “significantly lower level of expression” of a marker refers to an expression level in a test sample that is at least twice, and more preferably three, four, five or ten times lower than the expression level of the marker in a control sample (e.g., sample from a healthy subject not having the marker associated disease) and preferably, the average expression level of the marker in several control samples.

As used herein, the term “promoter/regulatory sequence” means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue-specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living human cell under most or all physiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living human cell substantially only when an inducer which corresponds to the promoter is present in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a living human cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

A “transcribed polynucleotide” or “nucleotide transcript” is a polynucleotide (e.g. an mRNA, hnRNA, a cDNA, or an analog of such RNA or cDNA) which is complementary to or homologous with all or a portion of a mature mRNA made by transcription of a marker of the invention and normal post-transcriptional processing (e.g. splicing), if any, of the RNA transcript, and reverse transcription of the RNA transcript.

“Complementary” refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds (“base pairing”) with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.

“Homologous” as used herein, refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. When a nucleotide residue position in both regions is occupied by the same nucleotide residue, then the regions are homologous at that position. A first region is homologous to a second region if at least one nucleotide residue position of each region is occupied by the same residue. Homology between two regions is expressed in terms of the proportion of nucleotide residue positions of the two regions that are occupied by the same nucleotide residue. By way of example, a region having the nucleotide sequence 5′-ATTGCC-3′ and a region having the nucleotide sequence 5′-TATGGC-3′ share 50% homology. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residue positions of each of the portions are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions of each of the portions are occupied by the same nucleotide residue.

A molecule is “fixed” or “affixed” to a substrate if it is covalently or non-covalently associated with the substrate such the substrate can be rinsed with a fluid (e.g. standard saline citrate, pH 7.4) without a substantial fraction of the molecule dissociating from the substrate.

As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in an organism found in nature.

A cancer is “inhibited” if at least one symptom of the cancer is alleviated, terminated, slowed, or prevented. As used herein, thymic lymphoma and hamartomatous tumors are also “inhibited” if recurrence or metastasis of the cancer is reduced, slowed, delayed, or prevented.

A kit is any manufacture (e.g. a package or container) comprising at least one reagent, e.g. a probe, for specifically detecting the expression of a marker of the invention. The kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention.

“Proteins of the invention” encompass marker proteins and their fragments; variant marker proteins and their fragments; peptides and polypeptides comprising an at least 15 amino acid segment of a marker or variant marker protein; and fusion proteins comprising a marker or variant marker protein, or an at least 15 amino acid segment of a marker or variant marker protein.

Unless otherwise specified here within, the terms “antibody” and “antibodies” broadly encompass naturally-occurring forms of antibodies (e.g. IgG, IgA, IgM, IgE) and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies and multi-specific antibodies, as well as fragments and derivatives of all of the foregoing, which fragments and derivatives have at least an antigenic binding site. Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody.

DESCRIPTION

The present invention is based, in part, on newly identified markers which are over-expressed or under-expressed in thymic lymphoma cells as compared to their expression in normal (i.e. non-cancerous) cells. The enhanced expression of one or more of these markers in thymic lymphoma cells is herein correlated with the cancerous state of the tissue. The invention provides compositions, kits, and methods for assessing the cancerous state of thymic lymphoma cells (e.g. cells obtained from a human, cultured human cells, archived or preserved human cells and in vivo cells) as well as treating patients afflicted with thymic lymphoma.

The present invention is also based, in part, on newly identified markers which are over-expressed or under-expressed in hamartomatous tumor cells as compared to their expression in normal (i.e. non-cancerous) cells. The enhanced expression of one or more of these markers in hamartomatous tumor cells is herein correlated with the cancerous state of the tissue. The invention provides compositions, kits, and methods for assessing the cancerous state of hamartomatous tumor cells (e.g. cells obtained from a human, cultured human cells, archived or preserved human cells and in vivo cells) as well as treating patients afflicted with hamartomatous tumors.

The compositions, kits, and methods of the invention have the following uses, among others:

1) assessing whether a patient is afflicted with thymic lymphoma or hamartomatous tumors;

2) assessing the stage of thymic lymphoma in a human patient;

3) assessing the grade of thymic lymphoma in a patient;

4) assessing the benign or malignant nature of thymic lymphoma in a patient;

5) assessing the metastatic potential of thymic lymphoma in a patient;

6) assessing the histological type of neoplasm associated with thymic lymphoma in a patient;

7) assessing the stage of the hamartomatous tumors in a human patient;

8) assessing the grade of the hamartomatous tumors in a patient;

9) assessing the benign or malignant nature of the hamartomatous tumors in a patient;

10) assessing the metastatic potential of the hamartomatous tumors in a patient;

11) assessing the histological type of neoplasm associated with the hamartomatous tumors in a patient;

12) making antibodies, antibody fragments or antibody derivatives that are useful for treating thymic lymphoma or hamartomatous tumors and/or assessing whether a patient is afflicted with thymic lymphoma or hamartomatous tumors;

13) assessing the presence of thymic lymphoma or hamartomatous tumor cells;

14) assessing the efficacy of one or more test compounds for inhibiting thymic lymphoma or hamartomatous tumors in a patient;

15) assessing the efficacy of a therapy for inhibiting thymic lymphoma or hamartomatous tumors in a patient;

16) monitoring the progression of thymic lymphoma or hamartomatous tumors in a patient;

17) selecting a composition or therapy for inhibiting thymic lymphoma or hamartomatous tumors in a patient;

18) treating a patient afflicted with thymic lymphoma or hamartomatous tumors;

19) inhibiting thymic lymphoma or hamartomatous tumors in a patient;

20) assessing the carcinogenic potential of a test compound;

21) preventing the onset of thymic lymphoma in a patient at risk for developing thymic lymphoma; and

22) preventing the onset of hamartomatous tumors in a patient at risk for developing hamartomatous tumors.

The invention thus includes a method of assessing whether a patient is afflicted with thymic lymphoma which includes assessing whether the patient has pre-metastasized thymic lymphoma. This method comprises comparing the level of expression of a marker of the invention (listed in Tables 1 and 2) in a patient sample and the normal level of expression of the marker in a control, e.g., a non-cancer sample. For the markers listed in Table 1, a significantly higher level of expression of the marker in the patient sample as compared to the normal level is an indication that the patient is afflicted with thymic lymphoma. For the markers listed in Table 2, a significantly lower level of expression of the marker in the patient sample as compared to the normal level is an indication that the patient is afflicted with thymic lymphoma.

The invention thus also includes a method of assessing whether a patient is afflicted with hamartomatous tumors which includes assessing whether the patient has pre-metastasized hamartomatous tumors. This method comprises comparing the level of expression of a marker of the invention (listed in Tables 3 and 4) in a patient sample and the normal level of expression of the marker in a control, e.g., a non-cancer sample. For the markers listed in Table 3, a significantly higher level of expression of the marker in the patient sample as compared to the normal level is an indication that the patient is afflicted with hamartomatous tumors. For the markers listed in Table 4, a significantly lower level of expression of the marker in the patient sample as compared to the normal level is an indication that the patient is afflicted with hamartomatous tumors.

Gene delivery vehicles, host cells and compositions (all described herein) containing nucleic acids comprising the entirety, or a segment of 15 or more nucleotides, of any of the nucleic acid sequences set forth in the Sequence Listing, or the complement of such sequences, and polypeptides comprising the entirety, or a segment of 10 or more amino acids, of any of the amino acid sequences set forth in the Sequence Listing, are also provided by this invention.

As described herein, thymic lymphoma or hamartomatous tumors in patients is associated with an increased or decreased level of expression of one or more markers of the invention. While, as discussed above, some of these changes in expression level result from occurrence of the thymic lymphoma or hamartomatous tumors, others of these changes induce, maintain, and promote the cancerous state of the thymic lymphoma or hamartomatous tumor cells. Thus, thymic lymphoma or hamartomatous tumors characterized by an increase or decrease in the level of expression of one or more markers of the invention can be inhibited by reducing or increasing and/or interfering with the expression of the markers and/or function of the proteins encoded by those markers.

Expression of a marker of the invention can be inhibited in a number of ways generally known in the art. For example, an antisense oligonucleotide can be provided to the thymic lymphoma or hamartomatous tumor cells in order to inhibit transcription, translation, or both, of the marker(s). Alternately, a polynucleotide encoding an antibody, an antibody derivative, or an antibody fragment which specifically binds a marker protein, and operably linked with an appropriate promoter/regulator region, can be provided to the cell in order to generate intracellular antibodies which will inhibit the function or activity of the protein. The expression and/or function of a marker may also be inhibited by treating the thymic lymphoma or hamartomatous tumor cell with an antibody, antibody derivative or antibody fragment that specifically binds a marker protein. Using the methods described herein, a variety of molecules, particularly including molecules sufficiently small that they are able to cross the cell membrane, can be screened in order to identify molecules which inhibit expression of a marker or inhibit the function of a marker protein. The compound so identified can be provided to the patient in order to inhibit thymic lymphoma or hamartomatous tumor cells of the patient.

Any marker or combination of markers of the invention, as well as any known markers in combination with the markers of the invention, may be used in the compositions, kits, and methods of the present invention. In general, it is preferable to use markers for which the difference between the level of expression of the marker in the thymic lymphoma or hamartomatous tumor cells and the level of expression of the same marker in normal cells is as great as possible. Although this difference can be as small as the limit of detection of the method for assessing expression of the marker, it is preferred that the difference be at least greater than the standard error of the assessment method, and preferably a difference of at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 100-, 500-, 1000-fold or greater than the level of expression of the same marker in normal tissue.

It is recognized that certain marker proteins are secreted from thymic lymphoma or hamartomatous tumor cells (i.e. one or both of normal and cancerous cells) to the extracellular space surrounding the cells. These markers may be used in certain embodiments of the compositions, kits, and methods of the invention, owing to the fact that such marker proteins can be detected in an associated body fluid sample, which may be more easily collected from a human patient than a tissue biopsy sample. Additional in vivo techniques for detection of a marker protein include introducing into a subject a labeled antibody directed against the protein. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

It is a simple matter for the skilled artisan to determine whether any particular marker protein is a secreted protein. In order to make this determination, the marker protein is expressed in, for example, a mammalian cell, extracellular fluid is collected, and the presence or absence of the protein in the extracellular fluid is assessed (e.g. using a labeled antibody which binds specifically with the protein).

The following is an example of a method which can be used to detect secretion of a protein. About 8×10¹⁰ 293 T cells are incubated at 37° C. in wells containing growth medium (Dulbecco's modified Eagle's medium {DMEM} supplemented with 10% fetal bovine serum) under a 5% (v/v) CO₂, 95% air atmosphere to about 60-70% confluence. The cells are then transfected using a standard transfection mixture comprising 2 micrograms of DNA comprising an expression vector encoding the protein and 10 microliters of LipofectAMINETm (GIBCO/BRL Catalog no. 18342-012) per well. The transfection mixture is maintained for about 5 hours, and then replaced with fresh growth medium and maintained in an air atmosphere. Each well is gently rinsed twice with DMEM which does not contain methionine or cysteine (DMEM-MC; ICN Catalog no. 16-424-54). About 1 milliliter of DMEM-MC and about 50 microcuries of Trans-35TM reagent (ICN Catalog no. 51006) are added to each well. The wells are maintained under the 5% CO₂ atmosphere described above and incubated at 37° C. for a selected period. Following incubation, 150 microliters of conditioned medium is removed and centrifuged to remove floating cells and debris. The presence of the protein in the supernatant is an indication that the protein is secreted.

It will be appreciated that patient samples containing thymic lymphoma or hamartomatous tumor cells may be used in the methods of the present invention. In these embodiments, the level of expression of the marker can be assessed by assessing the amount (e.g. absolute amount or concentration) of the marker in a thymic lymphoma or hamartomatous tumor cell sample, e.g., obtained from a patient. The cell sample can, of course, be subjected to a variety of well-known post-collection preparative and storage techniques (e.g., nucleic acid and/or protein extraction, fixation, storage, freezing, ultrafiltration, concentration, evaporation, centrifugation, etc.) prior to assessing the amount of the marker in the sample. Likewise, patient samples may also be subjected to post-collection preparative and storage techniques, e.g., fixation.

The compositions, kits, and methods of the invention can be used to detect expression of marker proteins having at least one portion which is displayed on the surface of cells which express it. It is a simple matter for the skilled artisan to determine whether a marker protein, or a portion thereof, is exposed on the cell surface. For example, immunological methods may be used to detect such proteins on whole cells, or well known computer-based sequence analysis methods may be used to predict the presence of at least one extracellular domain (i.e. including both secreted proteins and proteins having at least one cell-surface domain). Expression of a marker protein having at least one portion which is displayed on the surface of a cell which expresses it may be detected without necessarily lysing the cell (e.g. using a labeled antibody which binds specifically with a cell-surface domain of the protein).

Expression of a marker of the invention may be assessed by any of a wide variety of well known methods for detecting expression of a transcribed nucleic acid or protein. Non-limiting examples of such methods include immunological methods for detection of secreted, cell-surface, cytoplasmic, or nuclear proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods.

In one embodiment, expression of a marker is assessed using an antibody (e.g. a radio-labeled, chromophore-labeled, fluorophore-labeled, or enzyme-labeled antibody), an antibody derivative (e.g. an antibody conjugated with a substrate or with the protein or ligand of a protein-ligand pair {e.g. biotin-streptavidin}), or an antibody fragment (e.g. a single-chain antibody, an isolated antibody hypervariable domain, etc.) which binds specifically with a marker protein or fragment thereof, including a marker protein which has undergone all or a portion of its normal post-translational modification.

In another embodiment, expression of a marker is assessed by preparing mRNA/cDNA (i.e. a transcribed polynucleotide) from cells in a patient sample, and by hybridizing the mRNA/cDNA with a reference polynucleotide which is a complement of a marker nucleic acid, or a fragment thereof. cDNA can, optionally, be amplified using any of a variety of polymerase chain reaction methods prior to hybridization with the reference polynucleotide; preferably, it is not amplified. Expression of one or more markers can likewise be detected using quantitative PCR to assess the level of expression of the marker(s). Alternatively, any of the many known methods of detecting mutations or variants (e.g. single nucleotide polymorphisms, deletions, etc.) of a marker of the invention may be used to detect occurrence of a marker in a patient.

In a related embodiment, a mixture of transcribed polynucleotides obtained from the sample is contacted with a substrate having fixed thereto a polynucleotide complementary to or homologous with at least a portion (e.g. at least 7, 10, 15, 20, 25, 30, 40, 50, 100, 500, or more nucleotide residues) of a marker nucleic acid. If polynucleotides complementary to or homologous with are differentially detectable on the substrate (e.g. detectable using different chromophores or fluorophores, or fixed to different selected positions), then the levels of expression of a plurality of markers can be assessed simultaneously using a single substrate (e.g. a “gene chip” microarray of polynucleotides fixed at selected positions). When a method of assessing marker expression is used which involves hybridization of one nucleic acid with another, the hybridization may be performed under stringent hybridization conditions.

Because the compositions, kits, and methods of the invention rely on detection of a difference in expression levels of one or more markers of the invention, it is preferable that the level of expression of the marker is significantly greater than the minimum detection limit of the method used to assess expression in at least one of normal cells and cancerous thymic lymphoma or hamartomatous tumor cells.

It is understood that by routine screening of additional patient samples using one or more of the markers of the invention, it will be realized that certain of the markers are over-expressed in cancers of various types, including specific thymic lymphomas and hamartomatous tumors, as well as other lymphomas and conditions producing hamartomatous tumors. For example, it will be confirmed that some of the markers of the invention are over-expressed in most (i.e. 50% or more) or substantially all (i.e. 80% or more) of thymic lymphoma cells or hamartomatous tumor cells. Furthermore, it may be confirmed that certain of the markers of the invention are associated with lymphoma or hamartomatous tumors of various types including Mature B Cell Neoplasms (e.g. Chronic lymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocytic leukemia, Lymphoplasmacytic lymphoma/Waldenström macroglobulinemia, Splenic marginal zone lymphoma, Plasma cell neoplasms, Extranodal marginal zone B cell lymphoma (MALT lymphoma), Nodal marginal zone B cell lymphoma, Follicular lymphoma, Mantle cell lymphoma, Diffuse large B cell lymphoma, Mediastinal (thymic) large B cell lymphoma, Intravascular large B cell lymphoma, Primary effusion lymphoma, Burkitt lymphoma/leukemia, Lymphomatoid granulomatosis), Mature T cell and Natural Killer (NK) Cell Neoplasms (e.g. T cell prolymphocytic leukemia, T cell large granular lymphocytic leukemia, Aggressive NK cell leukemia, Adult T cell leukemia/lymphoma, Nasal type extranodal NK/T cell lymphoma, Enteropathy-type T cell lymphoma, Hepatosplenic T cell lymphoma, Blastic NK cell lymphoma, Mycosis fungoides/Sezary syndrome, Primary cutaneous CD30-positive T cell lymphoproliferative disorders, Angioimmunoblastic T cell lymphoma, Unspecified peripheral T cell lymphoma, Anaplastic large cell lymphoma), Hodgkin lymphoma (e.g. Nodular lymphocyte-predominant Hodgkin lymphoma, Classical Hodgkin lymphoma), Immunodeficiency-Associated Lymphoproliferative Disorders (i.e. those associated with a primary immune disorder, those associated with the Human Immunodeficiency Virus (HIV), those associated with Methotrexate therapy, those associated with organ transplantation), Histiocytic and Dendritic Cell Neoplasms (e.g. Histiocytic sarcoma, Langerhans cell histiocytosis, Langerhans cell sarcoma, Interdigitating dendritic cell sarcoma/tumour, Follicular dendritic cell sarcoma/tumour, Unspecified dendritic cell sarcoma), thymomas, hamartomatous tumors arising in a vascular organ (e.g. lung, liver, uterus, hypothalmus, kidney, spleen, skeletal muscle, abdominal wall, adrenal gland, bone marrow, omentum, lymph node, skin), malignant angiosarcomas derived from such hamartomatous tumors, hemangiomas, Cowden syndrome, Lhermitte-Duclos disease, and Bannayan-Zonana syndrome.

In addition, as a greater number of patient samples are assessed for expression of the markers of the invention and the outcomes of the individual patients from whom the samples were obtained are correlated, it will also be confirmed that altered expression of certain of the markers of the invention are strongly correlated with malignant cancers and that altered expression of other markers of the invention are strongly correlated with benign tumors. The compositions, kits, and methods of the invention are thus useful for characterizing one or more of the stage, grade, histological type, and benign/malignant nature of thymic lymphoma or hamartomatous tumors in patients.

When the compositions, kits, and methods of the invention are used for characterizing one or more of the stage, grade, histological type, and benign/malignant nature of thymic lymphoma or hamartomatous tumors in a patient, the marker or panel of markers of the invention may be selected such that a positive result is obtained in at least about 20%, or at least about 40%, 60%, or 80%, or in substantially all patients afflicted with thymic lymphoma or hamartomatous tumors of the corresponding stage, grade, histological type, or benign/malignant nature. The marker or panel of markers of the invention may also be selected such that a positive predictive value (PPV) of greater than about 10% is obtained for the general population (more preferably coupled with an assay specificity greater than 80%).

When a plurality of markers of the invention are used in the compositions, kits, and methods of the invention, the level of expression of each marker in a patient sample can be compared with the normal level of expression of each of the plurality of markers in non-cancerous samples of the same type, either in a single reaction mixture (i.e. using reagents, such as different fluorescent probes, for each marker) or in individual reaction mixtures corresponding to one or more of the markers. In one embodiment, a significantly increased level of expression of more than one of the plurality of markers in the sample, relative to the corresponding normal levels, is an indication that the patient is afflicted with thymic lymphoma or hamartomatous tumors. In another embodiment, a significantly decreased level of expression of more than one of the plurality of markers in the sample, relative to the corresponding normal levels, is an indication that the patient is afflicted with thymic lymphoma or hamartomatous tumors. When a plurality of markers is used, it is preferred that 2, 3, 4, 5, 8, 10, 12, 15, 20, 30, or 50 or more individual markers be used, wherein fewer markers are preferred.

In order to maximize the sensitivity of the compositions, kits, and methods of the invention, it is preferable that the marker of the invention used therein be a marker which has a restricted tissue distribution, e.g., normally not expressed in tissues unaffected by thymic lymphoma or hamartomatous tumors.

Markers known to be associated with thymic lymphoma or hamartomatous tumors are not, of course, included among the markers of the invention, although they may be used together with one or more markers of the invention in a panel of markers, for example. It is well known that certain types of genes, such as oncogenes, tumor suppressor genes, growth factor-like genes, protease-like genes, and protein kinase-like genes are often involved with development of cancers of various types. Thus, among the markers of the invention, use of those which correspond to proteins which resemble known proteins encoded by known oncogenes and tumor suppressor genes, and those which correspond to proteins which resemble growth factors, proteases, and protein kinases may be preferred.

It is recognized that the compositions, kits, and methods of the invention will be of particular utility to patients having an enhanced risk of developing thymic lymphoma or hamartomatous tumors and their medical advisors. Patients recognized as having an enhanced risk of developing thymic lymphoma or hamartomatous tumors include, for example, patients having a familial history of thymic lymphoma or hamartomatous tumors, patients identified as having a mutant oncogene (i.e. at least one allele), and patients of advancing age (i.e. individuals older than about 50 or 60 years).

The level of expression of a marker in normal (i.e. non-cancerous) human tissue can be assessed in a variety of ways. In one embodiment, this normal level of expression is assessed by assessing the level of expression of the marker in a portion of cells which appears to be non-cancerous and by comparing this normal level of expression with the level of expression in a portion of the cells which is suspected of being cancerous. Alternately, and particularly as further information becomes available as a result of routine performance of the methods described herein, population-average values for normal expression of the markers of the invention may be used. In other embodiments, the ‘normal’ level of expression of a marker may be determined by assessing expression of the marker in a patient sample obtained from a non-cancer-afflicted patient, from a patient sample obtained from a patient before the suspected onset of thymic lymphoma or hamartomatous tumors in the patient, from archived patient samples, and the like.

The invention includes compositions, kits, and methods for assessing the presence of thymic lymphoma or hamartomatous tumor cells in a sample (e.g. an archived tissue sample or a sample obtained from a patient). These compositions, kits, and methods are substantially the same as those described above, except that, where necessary, the compositions, kits, and methods are adapted for use with samples other than patient samples. For example, when the sample to be used is a parafinized, archived human tissue sample, it can be necessary to adjust the ratio of compounds in the compositions of the invention, in the kits of the invention, or the methods used to assess levels of marker expression in the sample. Such methods are well known in the art and within the skill of the ordinary artisan.

The invention includes a kit for assessing the presence of thymic lymphoma or hamartomatous tumor cells (e.g. in a sample such as a patient sample). The kit comprises a plurality of reagents, each of which is capable of binding specifically with a marker nucleic acid or protein. Suitable reagents for binding with a marker protein include antibodies, antibody derivatives, antibody fragments, and the like. Suitable reagents for binding with a marker nucleic acid (e.g. a genomic DNA, an mRNA, a spliced mRNA, a cDNA, or the like) include complementary nucleic acids. For example, the nucleic acid reagents may include oligonucleotides (labeled or non-labeled) fixed to a substrate, labeled oligonucleotides not bound with a substrate, pairs of PCR primers, molecular beacon probes, and the like.

The kit of the invention may optionally comprise additional components useful for performing the methods of the invention. By way of example, the kit may comprise fluids (e.g. SSC buffer) suitable for annealing complementary nucleic acids or for binding an antibody with a protein with which it specifically binds, one or more sample compartments, an instructional material which describes performance of a method of the invention, a sample of normal cells, a sample of thymic lymphoma or hamartomatous tumor cells, and the like.

The invention also includes a method of making an isolated hybridoma which produces an antibody useful for assessing whether patient is afflicted with thymic lymphoma or hamartomatous tumors. In this method, a protein or peptide comprising the entirety or a segment of a marker protein is synthesized or isolated (e.g. by purification from a cell in which it is expressed or by transcription and translation of a nucleic acid encoding the protein or peptide in vivo or in vitro using known methods). A vertebrate, preferably a mammal such as a mouse, rat, rabbit, or sheep, is immunized using the protein or peptide. The vertebrate may optionally (and preferably) be immunized at least one additional time with the protein or peptide, so that the vertebrate exhibits a robust immune response to the protein or peptide. Splenocytes are isolated from the immunized vertebrate and fused with an immortalized cell line to form hybridomas, using any of a variety of methods well known in the art. Hybridomas formed in this manner are then screened using standard methods to identify one or more hybridomas which produce an antibody which specifically binds with the marker protein or a fragment thereof. The invention also includes hybridomas made by this method and antibodies made using such hybridomas.

The invention also includes a method of assessing the efficacy of a test compound for inhibiting thymic lymphoma or hamartomatous tumor cells. As described above, differences in the level of expression of the markers of the invention correlate with the cancerous state of thymic lymphoma or hamartomatous tumor cells. Although it is recognized that changes in the levels of expression of certain of the markers of the invention likely result from the cancerous state of thymic lymphoma or hamartomatous tumor cells, it is likewise recognized that changes in the levels of expression of other of the markers of the invention induce, maintain, and promote the cancerous state of those cells. Thus, compounds which inhibit thymic lymphoma or hamartomatous tumors in a patient will cause the level of expression of one or more of the markers of the invention to change to a level nearer the normal level of expression for that marker (i.e. the level of expression for the marker in non-cancerous cells).

This method thus comprises comparing expression of a marker in a first thymic lymphoma or hamartomatous tumor cell sample and maintained in the presence of the test compound and expression of the marker in a second thymic lymphoma or hamartomatous tumor cell sample and maintained in the absence of the test compound. For the markers provided in Tables 1 and 3, a significantly reduced expression of a marker of the invention in the presence of the test compound is an indication that the test compound inhibits thymic lymphoma or hamartomatous tumors, respectively. For the markers provided in Tables 2 and 4, a significantly increased expression of a marker of the invention in the presence of the test compound is an indication that the test compound inhibits thymic lymphoma or hamartomatous tumors, respectively. The thymic lymphoma or hamartomatous tumor cell samples may, for example, be aliquots of a single sample of normal cells obtained from a patient, pooled samples of normal cells obtained from a patient, cells of a normal cell line, aliquots of a single sample of thymic lymphoma or hamartomatous tumor cancer cells obtained from a patient, pooled samples of thymic lymphoma or hamartomatous tumor cancer cells obtained from a patient, cells of a thymic lymphoma or hamartomatous tumor cancer cell line, or the like. In one embodiment, the samples are thymic lymphoma or hamartomatous tumor cancer cells obtained from a patient and a plurality of compounds known to be effective for inhibiting various cancers are tested in order to identify the compound which is likely to best inhibit the thymic lymphoma or hamartomatous tumors in the patient.

This method may likewise be used to assess the efficacy of a therapy for inhibiting thymic lymphoma or hamartomatous tumors in a patient. In this method, the level of expression of one or more markers of the invention in a pair of samples (one subjected to the therapy, the other not subjected to the therapy) is assessed. As with the method of assessing the efficacy of test compounds, if the therapy induces a significantly lower level of expression of a marker provided in Table 1 or 3, then the therapy is efficacious for inhibiting thymic lymphoma or hamartomatous tumors, respectively. Likewise, if the therapy induces a significantly higher of expression of a marker provided in Table 2 or 4, then the therapy is efficacious for inhibiting thymic lymphoma or hamartomatous tumors, respectively. As above, if samples from a selected patient are used in this method, then alternative therapies can be assessed in vitro in order to select a therapy most likely to be efficacious for inhibiting thymic lymphoma or hamartomatous tumors in the patient.

As described above, the cancerous state of human thymic lymphoma and hamartomatous tumor cells is correlated with changes in the levels of expression of the markers of the invention. The invention includes a method for assessing the human cell carcinogenic potential of a test compound. This method comprises maintaining separate aliquots of human cells in the presence and absence of the test compound. Expression of a marker of the invention in each of the aliquots is compared. For the markers provided in Tables 1 and 3, a significantly higher level of expression of a marker of the invention in the aliquot maintained in the presence of the test compound (relative to the aliquot maintained in the absence of the test compound) is an indication that the test compound possesses human cell carcinogenic potential. For the markers provided in Tables 2 and 4, a significantly lower level of expression of a marker of the invention in the aliquot maintained in the presence of the test compound (relative to the aliquot maintained in the absence of the test compound) is an indication that the test compound possesses human cell carcinogenic potential. The relative carcinogenic potentials of various test compounds can be assessed by comparing the degree of enhancement or inhibition of the level of expression of the relevant markers, by comparing the number of markers for which the level of expression is enhanced or inhibited, or by comparing both.

In another aspect, the invention relates to the use of FoxO target genes (Tables 1-4, 6 and 7) in normalizing the vascular endothelial cells by regulating their proliferation and apoptosis in the diabetic retina. The invention further provides a method of treating a diabetic patient in need of treatment for the retinopathy comprising modulating the expression level of FoxO target genes other than FoxO itself for proliferative diabetic retinopathy (PDR).

Diabetic retinopathy is the most common and often serious eye complication for diabetic patients. In fact, nearly half of the individuals suffering from diabetes suffer from poor vision or even blindness. During the development of diabetes, elevated blood glucose levels can damage blood vessels causing leakage of blood and serum into the retina. In turn, insufficient oxygen and nutrient supply in the retina by dysfunctional blood vessels triggers proliferative diabetic retinopathy where abnormal and chaotically oriented new blood vessels frow on the retina. Not only do these vessels fail to nourish the retina propertly, they further pose serious risks to vision: e.g., exudates into the vitreous cavity may block light from reaching the retina, scar tissue formation may lead to separation of the retina from the inside of the eye, and the formation of abnormal blood vessels on the iris may block the flow of fluid through the front portion of the eye elevating intraorbital pressure. Thus, inhibition of uncontrolled growth of endothelial cells in the retina may be used to treat proliferative diabetic retinopathy.

Excessive endothelial cell proliferation is often in response to VEGF (Ferrara, N. Endocr Rev. 25:581-611 (2004)). Clinically, intravitreal bevacizumab (humanized anti-VEGF-A monoclonal antibody, Avastin, rhuMAb-VEGF; Genentech, South San Francisco, Calif.), which neutralizes VEGF activity, is used in the treatment of proliferative diabetic retinopathy with complete resolution of angiographic leaking in short term therapy (Avery et al., Ophthalmology 113:1695 (2006); Freeman and Falkenstein, Retina, 26:853-858 (2006); Mason, J. O. et al., Am. J. Opthalmol. 142:685-688 (2006)). VEGF regulates the phosphorylation, subcellular localization, and thereby transcriptional activity of FoxOs by a PI3K-Akt signaling pathway, thus inhibiting endothelial cell apoptosis, increasing DNA synthesis, and decreasing expression of the cyclin-dependent kinase inhibitor (i.e. p27Kip) (Abid et al., Arteriosclerosis, Thromosis, and Vascular Biology 24:294-300 (2004)). Furthermore, FoxOs regulate the genes previously implicated in vascular remodeling and postnatal angiogenesis including eNOS and angiopoetins those are important mediators of VEGF-induced angiogenesis (Joussen et al., Am. J. Pathology, 160:1683-1693 (2002); Potente et al., J. Clin. Invest. 115:2382-92 (2005); Suganthalakshmi et al., Molecular Vision 12:336-341 (2006)).

As described herein, a method for treating a diabetic patient in need of treatment for retinopathy comprises increasing FoxO transcriptional activity and/or target gene modulation. Selective regulation of genes has been shown to be important for the proliferating endothelial cells. In one embodiment, suitable targets include genes differentially regulated by FoxO deletion in the affected vascular endothelial cells (Tables 3-4 and 6). In another embodiment, single or combination of siRNAs against those upregulated genes and/or cDNA expression of downregulated genes by viral delivery into the affected retina via a pharmaceutically acceptable form may be effective in resolution of proliferative diabetic retinopathy.

Various aspects of the invention are described in further detail in the following subsections.

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid molecules, including nucleic acids which encode a marker protein or a portion thereof. Isolated nucleic acids of the invention also include nucleic acid molecules sufficient for use as hybridization probes to identify marker nucleic acid molecules, and fragments of marker nucleic acid molecules, e.g., those suitable for use as PCR primers for the amplification or mutation of marker nucleic acid molecules. As used herein, the term “nucleic acid molecule” is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

An “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Preferably, an “isolated” nucleic acid molecule is free of sequences (preferably protein-encoding sequences) which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kB, 4 kB, 3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a CDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the present invention can be isolated using standard molecular biology techniques and the sequence information in the database records described herein. Using all or a portion of such nucleic acid sequences, nucleic acid molecules of the invention can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al., ed., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

A nucleic acid molecule of the invention can be amplified using cDNA, mRNA, or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, nucleotides corresponding to all or a portion of a nucleic acid molecule of the invention can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which has a nucleotide sequence complementary to the nucleotide sequence of a marker nucleic acid or to the nucleotide sequence of a nucleic acid encoding a marker protein. A nucleic acid molecule which is complementary to a given nucleotide sequence is one which is sufficiently complementary to the given nucleotide sequence that it can hybridize to the given nucleotide sequence thereby forming a stable duplex.

Moreover, a nucleic acid molecule of the invention can comprise only a portion of a nucleic acid sequence, wherein the full length nucleic acid sequence comprises a marker nucleic acid or which encodes a marker protein. Such nucleic acids can be used, for example, as a probe or primer. The probe/primer typically is used as one or more substantially purified oligonucleotides. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 7, preferably about 15, more preferably about 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or 400 or more consecutive nucleotides of a nucleic acid of the invention.

Probes based on the sequence of a nucleic acid molecule of the invention can be used to detect transcripts or genomic sequences corresponding to one or more markers of the invention. The probe comprises a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as part of a diagnostic test kit for identifying cells or tissues which misexpress the protein, such as by measuring levels of a nucleic acid molecule encoding the protein in a sample of cells from a subject, e.g., detecting mRNA levels or determining whether a gene encoding the protein has been mutated or deleted.

The invention further encompasses nucleic acid molecules that differ, due to degeneracy of the genetic code, from the nucleotide sequence of nucleic acids encoding a marker protein (e.g., a protein having one of the amino acid sequences set forth in the Sequence Listing), and thus encode the same protein.

It will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequence can exist within a population (e.g., the human population). Such genetic polymorphisms can exist among individuals within a population due to natural allelic variation. An allele is one of a group of genes which occur alternatively at a given genetic locus. In addition, it will be appreciated that DNA polymorphisms that affect RNA expression levels can also exist that may affect the overall expression level of that gene (e.g., by affecting regulation or degradation).

As used herein, the phrase “allelic variant” refers to a nucleotide sequence which occurs at a given locus or to a polypeptide encoded by the nucleotide sequence.

As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide corresponding to a marker of the invention. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of a given gene. Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals. This can be readily carried out by using hybridization probes to identify the same genetic locus in a variety of individuals. Any and all such nucleotide variations and resulting amino acid polymorphisms or variations that are the result of natural allelic variation and that do not alter the functional activity are intended to be within the scope of the invention.

In another embodiment, an isolated nucleic acid molecule of the invention is at least 7, 15, 20, 25, 30, 40, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 550, 650, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3500, 4000, 4500, or more nucleotides in length and hybridizes under stringent conditions to a marker nucleic acid or to a nucleic acid encoding a marker protein. As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% (65%, 70%, preferably 75%) identical to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in sections 6.3.1-6.3.6 of Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989). A non-limiting example of stringent hybridization conditions are hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C.

In addition to naturally-occurring allelic variants of a nucleic acid molecule of the invention that can exist in the population, the skilled artisan will further appreciate that sequence changes can be introduced by mutation thereby leading to changes in the amino acid sequence of the encoded protein, without altering the biological activity of the protein encoded thereby. For example, one can make nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues. A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence without altering the biological activity, whereas an “essential” amino acid residue is required for biological activity. For example, amino acid residues that are not conserved or only semi-conserved among homologs of various species may be non-essential for activity and thus would be likely targets for alteration. Alternatively, amino acid residues that are conserved among the homologs of various species (e.g., murine and human) may be essential for activity and thus would not be likely targets for alteration.

Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding a variant marker protein that contain changes in amino acid residues that are not essential for activity. Such variant marker proteins differ in amino acid sequence from the naturally-occurring marker proteins, yet retain biological activity. In one embodiment, such a variant marker protein has an amino acid sequence that is at least about 40% identical, 50%, 60%, 70%, 80%, 90%, 95%, or 98% identical to the amino acid sequence of a marker protein.

An isolated nucleic acid molecule encoding a variant marker protein can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of marker nucleic acids, such that one or more amino acid residue substitutions, additions, or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

The present invention encompasses antisense nucleic acid molecules, i.e., molecules which are complementary to a sense nucleic acid of the invention, e.g., complementary to the coding strand of a double-stranded marker cDNA molecule or complementary to a marker mRNA sequence. Accordingly, an antisense nucleic acid of the invention can hydrogen bond to (i.e. anneal with) a sense nucleic acid of the invention. The antisense nucleic acid can be complementary to an entire coding strand, or to only a portion thereof, e.g., all or part of the protein coding region (or open reading frame). An antisense nucleic acid molecule can also be antisense to all or part of a non-coding region of the coding strand of a nucleotide sequence encoding a marker protein. The non-coding regions (“5′ and 3′ untranslated regions”) are the 5′ and 3′ sequences which flank the coding region and are not translated into amino acids.

An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxyhnethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiour-acil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been sub-cloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a marker protein to thereby inhibit expression of the marker, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. Examples of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site or infusion of the antisense nucleic acid into an ovary-associated body fluid. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter may be used.

An antisense nucleic acid molecule of the invention can be an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual α-units, the strands run parallel to each other (Gaultier et al., 1987, Nucleic Acids Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al., 1987, Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et aL, 1987, FEBS Lett. 215:327-330).

The invention also encompasses ribozymes. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes as described in Haselhoff and Gerlach, 1988, Nature 334:585-591) can be used to catalytically cleave mRNA transcripts to thereby inhibit translation of the protein encoded by the mRNA. A ribozyme having specificity for a nucleic acid molecule encoding a marker protein can be designed based upon the nucleotide sequence of a cDNA corresponding to the marker. For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved (see Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742). Alternatively, an mRNA encoding a polypeptide of the invention can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (see, e.g., Bartel and Szostak, 1993, Science 261:1411-1418).

The invention also encompasses nucleic acid molecules which form triple helical structures. For example, expression of a marker of the invention can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the gene encoding the marker nucleic acid or protein (e.g., the promoter and/or enhancer) to form triple helical structures that prevent transcription of the gene in target cells. See generally Helene (1991) Anticancer Drug Des. 6(6):569-84; Helene (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14(12):807-15.

In various embodiments, the nucleic acid molecules of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al., 1996, Bioorganic & Medicinal Chemistry 4(1): 5-23). As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93:14670-675.

PNAs can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., Si nucleases (Hyrup (1996), supra; or as probes or primers for DNA sequence and hybridization (Hyrup, 1996, supra; Perry-O'Keefe et al., 1996, Proc. Natl. Acad. Sci. USA 93:14670-675).

In another embodiment, PNAs can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras can be generated which can combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup, 1996, supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996), supra, and Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs. Compounds such as 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite can be used as a link between the PNA and the 5′ end of DNA (Mag et al., 1989, Nucleic Acids Res. 17:5973-88). PNA monomers are then coupled in a step-wise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment (Finn et al., 1996, Nucleic Acids Res. 24(17):3357-63). Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment (Peterser et al., 1975, Bioorganic Med. Chem. Lett. 5:1119-11124).

In other embodiments, the oligonucleotide can include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al., 1988, Bio/Techniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, the oligonucleotide can be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

The invention also includes molecular beacon nucleic acids having at least one region which is complementary to a nucleic acid of the invention, such that the molecular beacon is useful for quantitating the presence of the nucleic acid of the invention in a sample. A “molecular beacon” nucleic acid is a nucleic acid comprising a pair of complementary regions and having a fluorophore and a fluorescent quencher associated therewith. The fluorophore and quencher are associated with different portions of the nucleic acid in such an orientation that when the complementary regions are annealed with one another, fluorescence of the fluorophore is quenched by the quencher. When the complementary regions of the nucleic acid are not annealed with one another, fluorescence of the fluorophore is quenched to a lesser degree. Molecular beacon nucleic acids are described, for example, in U.S. Pat. No. 5,876,930.

II. Isolated Proteins and Antibodies

One aspect of the invention pertains to isolated marker proteins and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise antibodies directed against a marker protein or a fragment thereof. In one embodiment, the native marker protein can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, a protein or peptide comprising the whole or a segment of the marker protein is produced by recombinant DNA techniques. Alternative to recombinant expression, such protein or peptide can be synthesized chemically using standard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a “contaminating protein”). When the protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation. When the protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly such preparations of the protein have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the polypeptide of interest.

Biologically active portions of a marker protein include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the marker protein, which include fewer amino acids than the full length protein, and exhibit at least one activity of the corresponding full-length protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the corresponding full-length protein. A biologically active portion of a marker protein of the invention can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length. Moreover, other biologically active portions, in which other regions of the marker protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of the native form of the marker protein.

Acceptable marker proteins may be those encoded by nucleotide sequences comprising the sequence of any of the sequences set forth in the Sequence Listing. Other useful proteins are substantially identical (e.g., at least about 40%, preferably 50%, 60%, 70%, 80%, 90%, 95%, or 99%) to one of these sequences and retain the functional activity of the corresponding naturally-occurring marker protein yet differ in amino acid sequence due to natural allelic variation or mutagenesis.

To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g. gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=# of identical positions/total # of positions (e.g., overlapping positions) times 100). In one embodiment the two sequences are the same length.

The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTP program, score=50, wordlength=3 to obtain amino acid sequences homologous to a protein molecules of the invention. To obtain gapped alignments for comparison purposes, a newer version of the BLAST algorithm called Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, which is able to perform gapped local alignments for the programs BLASTN, BLASTP and BLASTX. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) can be used. See http://www.ncbi.nlm.nih.gov. Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, (1988) CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Yet another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm as described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448. When using the FASTA algorithm for comparing nucleotide or amino acid sequences, a PAM120 weight residue table can, for example, be used with a k-tuple value of 2.

The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted.

The invention also provides chimeric or fusion proteins comprising a marker protein or a segment thereof. As used herein, a “chimeric protein” or “fusion protein” comprises all or part (preferably a biologically active part) of a marker protein operably linked to a heterologous polypeptide (i.e., a polypeptide other than the marker protein). Within the fusion protein, the term “operably linked” is intended to indicate that the marker protein or segment thereof and the heterologous polypeptide are fused in-frame to each other. The heterologous polypeptide can be fused to the aminoterminus or the carboxyl-terminus of the marker protein or segment.

One useful fusion protein is a GST fusion protein in which a marker protein or segment is fused to the carboxyl terminus of GST sequences. Such fusion proteins can facilitate the purification of a recombinant polypeptide of the invention.

In another embodiment, the fusion protein contains a heterologous signal sequence at its amino terminus. For example, the native signal sequence of a marker protein can be removed and replaced with a signal sequence from another protein. For example, the gp67 secretory sequence of the baculovirus envelope protein can be used as a heterologous signal sequence (Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 1992). Other examples of eukaryotic heterologous signal sequences include the secretory sequences of melittin and human placental alkaline phosphatase (Stratagene; La Jolla, Calif.). In yet another example, useful prokaryotic heterologous signal sequences include the phoA secretory signal (Sambrook et aL., supra) and the protein A secretory signal (Pharmacia Biotech; Piscataway, N.J.).

In yet another embodiment, the fusion protein is an immunoglobulin fusion protein in which all or part of a marker protein is fused to sequences derived from a member of the immunoglobulin protein family. The immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ligand (soluble or membrane-bound) and a protein on the surface of a cell (receptor), to thereby suppress signal transduction in vivo. The immunoglobulin fusion protein can be used to affect the bioavailability of a cognate ligand of a marker protein. Inhibition of ligand/receptor interaction can be useful therapeutically, both for treating proliferative and differentiative disorders and for modulating (e.g. promoting or inhibiting) cell survival. Moreover, the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies directed against a marker protein in a subject, to purify ligands and in screening assays to identify molecules which inhibit the interaction of the marker protein with ligands.

Chimeric and fusion proteins of the invention can be produced by standard recombinant DNA techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see, e.g., Ausubel et al., supra). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the polypeptide of the invention.

A signal sequence can be used to facilitate secretion and isolation of marker proteins. Signal sequences are typically characterized by a core of hydrophobic amino acids which are generally cleaved from the mature protein during secretion in one or more cleavage events. Such signal peptides contain processing sites that allow cleavage of the signal sequence from the mature proteins as they pass through the secretory pathway. Thus, the invention pertains to marker proteins, fusion proteins or segments thereof having a signal sequence, as well as to such proteins from which the signal sequence has been proteolytically cleaved (i.e., the cleavage products). In one embodiment, a nucleic acid sequence encoding a signal sequence can be operably linked in an expression vector to a protein of interest, such as a marker protein or a segment thereof. The signal sequence directs secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed, and the signal sequence is subsequently or concurrently cleaved. The protein can then be readily purified from the extracellular medium by art recognized methods. Alternatively, the signal sequence can be linked to the protein of interest using a sequence which facilitates purification, such as with a GST domain.

The present invention also pertains to variants of the marker proteins. Such variants have an altered amino acid sequence which can function as either agonists (mimetics) or as antagonists. Variants can be generated by mutagenesis, e.g. discrete point mutation or truncation. An agonist can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of the protein. An antagonist of a protein can inhibit one or more of the activities of the naturally occurring form of the protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the protein of interest. Thus, specific biological effects can be elicited by treatment with a variant of limited function. Treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein can have fewer side effects in a subject relative to treatment with the naturally occurring form of the protein.

Variants of a marker protein which function as either agonists (mimetics) or as antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the protein of the invention for agonist or antagonist activity. In one embodiment, a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display). There are a variety of methods which can be used to produce libraries of potential variants of the marker proteins from a degenerate oligonucleotide sequence. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, 1983, Tetrahedron 39:3; Itakura et al., 1984, Annu. Rev. Biochem. 53:323; Itakura et al., 1984, Science 198:1056; Ike et al., 1983 Nucleic Acid Res. 11:477).

In addition, libraries of segments of a marker protein can be used to generate a variegated population of polypeptides for screening and subsequent selection of variant marker proteins or segments thereof. For example, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with Si nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes amino terminal and internal fragments of various sizes of the protein of interest.

Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify variants of a protein of the invention (Arkin and Yourvan, 1992, Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al., 1993, Protein Engineering 6(3):327-331).

Another aspect of the invention pertains to antibodies directed against a protein of the invention. In some embodiments, the antibodies specifically bind a marker protein or a fragment thereof. The terms “antibody” and “antibodies” as used interchangeably herein refer to immunoglobulin molecules as well as fragments and derivatives thereof that comprise an immunologically active portion of an immunoglobulin molecule, (i.e., such a portion contains an antigen binding site which specifically binds an antigen, such as a marker protein, e.g., an epitope of a marker protein). An antibody which specifically binds to a protein of the invention is an antibody which binds the protein, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains the protein. Examples of an immunologically active portion of an immunoglobulin molecule include, but are not limited to, single-chain antibodies (scAb), F(ab) and F(ab′)₂ fragments.

An isolated protein of the invention or a fragment thereof can be used as an immunogen to generate antibodies. The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments for use as immunogens. The antigenic peptide of a protein of the invention comprises at least 8 or more (e.g. 10, 15, 20, 30 or more) amino acid residues of the amino acid sequence of one of the proteins of the invention, and encompasses at least one epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein. Epitopes encompassed by the antigenic peptide may be regions that are located on the surface of the protein, e.g., hydrophilic regions. Hydrophobicity sequence analysis, hydrophilicity sequence analysis, or similar analyses can be used to identify hydrophilic regions. In some embodiments, an isolated marker protein or fragment thereof is used as an immunogen.

An immunogen typically is used to prepare antibodies by immunizing a suitable (i.e. immunocompetent) subject such as a rabbit, goat, mouse, or other mammal or vertebrate. An appropriate immunogenic preparation can contain, for example, recombinantly-expressed or chemically-synthesized protein or peptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulatory agent. Immunogen compositions may be those that contain no other human proteins such as, for example, immunogen compositions made using a non-human host cell for recombinant expression of a protein of the invention. In such a manner, the resulting antibody compositions have reduced or no binding of human proteins other than a protein of the invention.

The invention provides polyclonal and monoclonal antibodies. The term “monoclonal antibody” or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope. Polyclonal and monoclonal antibody compositions may be those that have been selected for antibodies directed against a protein of the invention. Polyclonal and monoclonal antibody preparations may also be those that contain only antibodies directed against a marker protein or fragment thereof.

Polyclonal antibodies can be prepared by immunizing a suitable subject with a protein of the invention as an immunogen. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide. At an appropriate time after immunization, e.g., when the specific antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies (mAb) by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497, the human B cell hybridoma technique (see Kozbor et al., 1983, Immunol. Today 4:72), the EBV-hybridoma technique (see Cole et al., pp. 77-96 In Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., 1985) or trioma techniques. The technology for producing hybridomas is well known (see generally Current Protocols in Immunology, Coligan et al. ed., John Wiley & Sons, N.Y., 1994). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide of interest, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody directed against a protein of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide of interest. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Pat. No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-734.

The invention also provides recombinant antibodies that specifically bind a protein of the invention. In some embodiments, the recombinant antibodies specifically bind a marker protein or fragment thereof. Recombinant antibodies include, but are not limited to, chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, single-chain antibodies and multi-specific antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. (See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397, which are incorporated herein by reference in their entirety.) Single-chain antibodies have an antigen binding site and consist of a single polypeptide. They can be produced by techniques known in the art, for example using methods described in Ladner et. al U.S. Pat. No. 4,946,778 (which is incorporated herein by reference in its entirety); Bird et al., (1988) Science 242:423-426; Whitlow et al., (1991) Methods in Enzymology 2:1-9; Whitlow et al., (1991) Methods in Enzymology 2:97-105; and Huston et al., (1991) Methods in Enzymology Molecular Design and Modeling: Concepts and Applications 203:46-88. Multi-specific antibodies are antibody molecules having at least two antigen-binding sites that specifically bind different antigens. Such molecules can be produced by techniques known in the art, for example using methods described in Segal, U.S. Pat. No. 4,676,980 (the disclosure of which is incorporated herein by reference in its entirety); Holliger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Whitlow et al., (1994) Protein Eng. 7:1017-1026 and U.S. Pat. No. 6,121,424.

Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. (See, e.g., Queen, U.S. Pat. No. 5,585,089, which is incorporated herein by reference in its entirety.) Humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication No. WO 87/02671; European Patent Application 184,187; European Patent Application 171,496; European Patent Application 173,494; PCT Publication No. WO 86/01533; U.S. Pat. No. 4,816,567; European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et aL. (1987) Cancer Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison (1985) Science 229:1202-1207; Oi et al. (1986) Bio/Techniques 4:214; U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141:4053-4060.

More particularly, humanized antibodies can be produced, for example, using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide corresponding to a marker of the invention. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995) Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No. 5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806. In addition, companies such as Abgenix, Inc. (Freemont, Calif.), can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a murine antibody, is used to guide the selection of a completely human antibody recognizing the same epitope (Jespers et al., 1994, Bio/technology 12:899-903).

The antibodies of the invention can be isolated after production (e.g., from the blood or serum of the subject) or synthesis and further purified by well-known techniques. For example, IgG antibodies can be purified using protein-A chromatography. Antibodies specific for a protein of the invention can be selected or (e.g., partially purified) or purified by, e.g., affinity chromatography. For example, a recombinantly expressed and purified (or partially purified) protein of the invention is produced as described herein, and covalently or non-covalently coupled to a solid support such as, for example, a chromatography column. The column can then be used to affinity purify antibodies specific for the proteins of the invention from a sample containing antibodies directed against a large number of different epitopes, thereby generating a substantially purified antibody composition, i.e., one that is substantially free of contaminating antibodies. By a substantially purified antibody composition is meant, in this context, that the antibody sample contains at most only 30% (by dry weight) of contaminating antibodies directed against epitopes other than those of the desired protein of the invention, and preferably at most 20%, yet more preferably at most 10%, and most preferably at most 5% (by dry weight) of the sample is contaminating antibodies. A purified antibody composition means that at least 99% of the antibodies in the composition are directed against the desired protein of the invention.

In one embodiment, the substantially purified antibodies of the invention may specifically bind to a signal peptide, a secreted sequence, an extracellular domain, a transmembrane or a cytoplasmic domain or cytoplasmic membrane of a protein of the invention. In another embodiment, the substantially purified antibodies of the invention specifically bind to a secreted sequence or an extracellular domain of the amino acid sequences of a protein of the invention. In still another embodiment, the substantially purified antibodies of the invention specifically bind to a secreted sequence or an extracellular domain of the amino acid sequences of a marker protein.

An antibody directed against a protein of the invention can be used to isolate the protein by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, such an antibody can be used to detect the marker protein or fragment thereof (e.g., in a cellular lysate or cell supernatant) in order to evaluate the level and pattern of expression of the marker. The antibodies can also be used diagnostically to monitor protein levels in tissues or body fluids as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by the use of an antibody derivative, which comprises an antibody of the invention coupled to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Antibodies of the invention may also be used as therapeutic agents in treating cancers. In one embodiment, completely human antibodies of the invention are used for therapeutic treatment of human cancer patients, particularly those having thymic lymphoma or hamartomatous tumors. In another embodiment, antibodies that bind specifically to a marker protein or fragment thereof are used for therapeutic treatment. Further, such therapeutic antibody may be an antibody derivative or immunotoxin comprising an antibody conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

The conjugated antibodies of the invention can be used for modifying a given biological response, for the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as ribosome-inhibiting protein (see Better et al., U.S. Pat. No. 6,146,631, the disclosure of which is incorporated herein in its entirety), abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62:119-58 (1982).

Accordingly, in one aspect, the invention provides substantially purified antibodies, antibody fragments and derivatives, all of which specifically bind to a protein of the invention and preferably, a marker protein. In various embodiments, the substantially purified antibodies of the invention, or fragments or derivatives thereof, can be human, non-human, chimeric and/or humanized antibodies. In another aspect, the invention provides non-human antibodies, antibody fragments and derivatives, all of which specifically bind to a protein of the invention and preferably, a marker protein. Such non-human antibodies can be goat, mouse, sheep, horse, chicken, rabbit, or rat antibodies. Alternatively, the non-human antibodies of the invention can be chimeric and/or humanized antibodies. In addition, the non-human antibodies of the invention can be polyclonal antibodies or monoclonal antibodies. In still a further aspect, the invention provides monoclonal antibodies, antibody fragments and derivatives, all of which specifically bind to a protein of the invention and preferably, a marker protein. The monoclonal antibodies can be human, humanized, chimeric and/or non-human antibodies.

The invention also provides a kit containing an antibody of the invention conjugated to a detectable substance, and instructions for use. Still another aspect of the invention is a pharmaceutical composition comprising an antibody of the invention. In one embodiment, the pharmaceutical composition comprises an antibody of the invention and a pharmaceutically acceptable carrier.

III. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a marker protein (or a portion of such a protein). As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors, namely expression vectors, are capable of directing the expression of genes to which they are operably linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors). However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell. This means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Methods in Enzymology: Gene Expression Technology vol. 185, Academic Press, San Diego, Calif. (1991). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.

The recombinant expression vectors of the invention can be designed for expression of a marker protein or a segment thereof in prokaryotic (e.g., E. coli) or eukaryotic cells (e.g., insect cells {using baculovirus expression vectors}, yeast cells or mammalian cells). Suitable host cells are discussed further in Goeddel, supra. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988, Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., 1988, Gene 69:301-315) and pET 11d (Studier et al., p. 60-89, In Gene Expression Technology: Methods in Enzymology vol. 1 85, Academic Press, San Diego, Calif., 1991). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 11d vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a co-expressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, p. 119-128, In Gene Expression Technology: Methods in Enzymology vol. 185, Academic Press, San Diego, Calif., 1990). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., 1992, Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

In another embodiment, the expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari et al., 1987, EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, 1982, Cell 30:933-943), pJRY88 (Schultz et al., 1987, Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and pPicZ (Invitrogen Corp, San Diego, Calif.).

Alternatively, the expression vector is a baculovirus expression vector. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., 1983, Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989, Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987, Nature 329:840) and pMT2PC (Kaufmnan et al., 1987, EMBO J. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook et al., supra.

In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al., 1987, Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton, 1988, Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989, EMBO J. 8:729-733) and immunoglobulins (Banedji et al., 1983, Cell 33:729-740; Queen and Baltimore, 1983, Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989, Proc. NatL. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al., 1985, Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss, 1990, Science 249:374-379) and the α-fetoprotein promoter (Camper and Tilghman, 1989, Genes Dev. 3:537-546).

The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operably linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to the mRNA encoding a polypeptide of the invention. Regulatory sequences operably linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue-specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid, or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub et al., 1986, Trends in Genetics, Vol. 1(1).

Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

A host cell can be any prokaryotic (e.g., E. coli) or eukaryotic cell (e.g., insect cells, yeast or mammalian cells).

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (supra), and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker will survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce a marker protein or a segment thereof. Accordingly, the invention further provides methods for producing a marker protein or a segment thereof using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of the invention (into which a recombinant expression vector encoding a marker protein or a segment thereof has been introduced) in a suitable medium such that the is produced. In another embodiment, the method further comprises isolating the marker protein or a segment thereof from the medium or the host cell.

The host cells of the invention can also be used to produce nonhuman transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which a sequences encoding a marker protein or a segment thereof have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous sequences encoding a marker protein of the invention have been introduced into their genome or homologous recombinant animals in which endogenous gene(s) encoding a marker protein have been altered. Such animals are useful for studying the function and/or activity of the marker protein and for identifying and/or evaluating modulators of marker protein. As used herein, a “transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, an “homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.

A transgenic animal of the invention can be created by introducing a nucleic acid encoding a marker protein into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the polypeptide of the invention to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, U.S. Pat. No. 4,873,191 and in Hogan, Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986. Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of mRNA encoding the transgene in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying the transgene can further be bred to other transgenic animals carrying other transgenes.

To create an homologous recombinant animal, a vector is prepared which contains at least a portion of a gene encoding a marker protein into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the gene. In one embodiment, the vector is designed such that, upon homologous recombination, the endogenous gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a “knock out” vector). Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous protein). In the homologous recombination vector, the altered portion of the gene is flanked at its 5′ and 3′ ends by additional nucleic acid of the gene to allow for homologous recombination to occur between the exogenous gene carried by the vector and an endogenous gene in an embryonic stem cell. The additional flanking nucleic acid sequences are of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5′ and 3′ ends) are included in the vector (see, e.g., Thomas and Capecchi, 1987, Cell 51:503 for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced gene has homologously recombined with the endogenous gene are selected (see, e.g., Li et al., 1992, Cell 69:915). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see, e.g., Bradley, Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, Ed., IRL, Oxford, 1987, pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley (1991) Current Opinion in Bio/Technology 2:823-829 and in PCT Publication NOS. WO 90/11354, WO 91/01140, WO 92/0968, and WO 93/04169.

In another embodiment, transgenic non-human animals can be produced which contain selected systems which allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al (1992) Proc. Natl. Acad. Sci. USA 89:6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al., 1991, Science 251:1351-1355). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al. (1997) Nature 385:810-813 and PCT Publication NOS. WO 97/07668 and WO 97/07669.

IV. Pharmaceutical Compositions

The nucleic acid molecules, polypeptides, and antibodies (also referred to herein as “active compounds”) of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

The invention includes methods for preparing pharmaceutical compositions for modulating the expression or activity of a marker nucleic acid or protein. Such methods comprise formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a marker nucleic acid or protein. Such compositions can further include additional active agents. Thus, the invention further includes methods for preparing a pharmaceutical composition by formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a marker nucleic acid or protein and one or more additional active compounds.

The invention also provides methods (also referred to herein as “screening assays”) for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, peptoids, small molecules or other drugs) which (a) bind to the marker, or (b) have a modulatory (e.g., stimulatory or inhibitory) effect on the activity of the marker or, more specifically, (c) have a modulatory effect on the interactions of the marker with one or more of its natural substrates (e.g., peptide, protein, hormone, co-factor, or nucleic acid), or (d) have a modulatory effect on the expression of the marker. Such assays typically comprise a reaction between the marker and one or more assay components. The other components may be either the test compound itself, or a combination of test compound and a natural binding partner of the marker.

The test compounds of the present invention may be obtained from any available source, including systematic libraries of natural and/or synthetic compounds. Test compounds may also be obtained by any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann et aL, 1994, J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the ‘one-bead one-compound’ library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, 1997, Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et aL (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten, 1992, Biotechniques 13:412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria and/or spores, (Ladner, U.S. Pat. No. 5,223,409), plasmids (Cull et al, 1992, Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al, 1990, Proc. Natl. Acad. Sci. 87:6378-6382; Felici, 1991, J. Mol. Biol. 222:301-310; Ladner, supra.).

In one embodiment, the invention provides assays for screening candidate or test compounds which are substrates of a protein encoded by or corresponding to a marker or biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds which bind to a protein encoded by or corresponding to a marker or biologically active portion thereof. Determining the ability of the test compound to directly bind to a protein can be accomplished, for example, by coupling the compound with a radioisotope or enzymatic label such that binding of the compound to the marker can be determined by detecting the labeled marker compound in a complex. For example, compounds (e.g., marker substrates) can be labeled with ¹²⁵I, ³⁵S or ¹⁴C, or ³H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, assay components can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

In another embodiment, the invention provides assays for screening candidate or test compounds which modulate the expression of a marker or the activity of a protein encoded by or corresponding to a marker, or a biologically active portion thereof. In all likelihood, the protein encoded by or corresponding to the marker can, in vivo, interact with one or more molecules, such as but not limited to, peptides, proteins, hormones, cofactors and nucleic acids. For the purposes of this discussion, such cellular and extracellular molecules are referred to herein as “binding partners” or marker “substrate”.

One necessary embodiment of the invention in order to facilitate such screening is the use of a protein encoded by or corresponding to marker to identify the protein's natural in vivo binding partners. There are many ways to accomplish this which are known to one skilled in the art. One example is the use of the marker protein as “bait protein” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al, 1993, Cell 72:223-232; Madura et al, 1993, J. Biol. Chem. 268:12046-12054; Bartel et al, 1993, Biotechniques 14:920-924; Iwabuchi et al, 1993 Oncogene 8:1693-1696; Brent WO94/10300) in order to identify other proteins which bind to or interact with the marker (binding partners) and, therefore, are possibly involved in the natural finction of the marker. Such marker binding partners are also likely to be involved in the propagation of signals by the marker protein or downstream elements of a marker protein-mediated signaling pathway. Alternatively, such marker protein binding partners may also be found to be inhibitors of the marker protein.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that encodes a marker protein fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact, in vivo, forming a marker-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be readily detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the marker protein.

In a further embodiment, assays may be devised through the use of the invention for the purpose of identifying compounds which modulate (e.g., affect either positively or negatively) interactions between a marker protein and its substrates and/or binding partners. Such compounds can include, but are not limited to, molecules such as antibodies, peptides, hormones, oligonucleotides, nucleic acids, and analogs thereof. Such compounds may also be obtained from any available source, including systematic libraries of natural and/or synthetic compounds. Assay components for use in this embodiment may be thymic lymphoma or hamartomatous tumor cell cancer marker proteins identified herein, the known binding partners and/or substrates of same, and the test compound. Test compounds can be supplied from any source.

The basic principle of the assay systems used to identify compounds that interfere with the interaction between the marker protein and its binding partner involves preparing a reaction mixture containing the marker protein and its binding partner under conditions and for a time sufficient to allow the two products to interact and bind, thus forming a complex. In order to test an agent for inhibitory activity, the reaction mixture is prepared in the presence and absence of the test compound. The test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of the marker protein and its binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the marker protein and its binding partner is then detected. The formation of a complex in the control reaction, but less or no such formation in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the marker protein and its binding partner. Conversely, the formation of more complex in the presence of compound than in the control reaction indicates that the compound may enhance interaction of the marker protein and its binding partner.

The assay for compounds that interfere with the interaction of the marker protein with its binding partner may be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the marker protein or its binding partner onto a solid phase and detecting complexes anchored to the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the marker proteins and the binding partners (e.g., by competition) can be identified by conducting the reaction in the presence of the test substance, i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the marker and its interactive binding partner. Alternatively, test compounds that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are briefly described below.

In a heterogeneous assay system, either the marker protein or its binding partner is anchored onto a solid surface or matrix, while the other corresponding non-anchored component may be labeled, either directly or indirectly. In practice, microtitre plates are often utilized for this approach. The anchored species can be immobilized by a number of methods, either non-covalent or covalent, that are typically well known to one who practices the art. Non-covalent attachment can often be accomplished simply by coating the solid surface with a solution of the marker protein or its binding partner and drying. Alternatively, an immobilized antibody specific for the assay component to be anchored can be used for this purpose. Such surfaces can often be prepared in advance and stored.

In related embodiments, a fusion protein can be provided which adds a domain that allows one or both of the assay components to be anchored to a matrix. For example, glutathione-S-transferase/marker fusion proteins or glutathione-S-transferase/binding partner can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed marker or its binding partner, and the mixture incubated under conditions conducive to complex formation (e.g., physiological conditions). Following incubation, the beads or microtiter plate wells are washed to remove any unbound assay components, the immobilized complex assessed either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of marker binding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either a marker protein or a marker protein binding partner can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated marker protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). In certain embodiments, the protein-immobilized surfaces can be prepared in advance and stored.

In order to conduct the assay, the corresponding partner of the immobilized assay component is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted assay components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds which modulate (inhibit or enhance) complex formation or which disrupt preformed complexes can be detected.

In an alternate embodiment of the invention, a homogeneous assay may be used. This is typically a reaction, analogous to those mentioned above, which is conducted in a liquid phase in the presence or absence of the test compound. The formed complexes are then separated from unreacted components, and the amount of complex fainted is determined. As mentioned for heterogeneous assay systems, the order of addition of reactants to the liquid phase can yield information about which test compounds modulate (inhibit or enhance) complex formation and which disrupt preformed complexes.

In such a homogeneous assay, the reaction products may be separated from unreacted assay components by any of a number of standard techniques, including but not limited to: differential centrifugation, chromatography, electrophoresis and immunoprecipitation. In differential centrifugation, complexes of molecules may be separated from uncomplexed molecules through a series of centrifugal steps, due to the different sedimentation equilibria of complexes based on their different sizes and densities (see, for example, Rivas, G., and Minton, A. P., Trends Biochem Sci 1993 August; 18(8):284-7). Standard chromatographic techniques may also be utilized to separate complexed molecules from uncomplexed ones. For example, gel filtration chromatography separates molecules based on size, and through the utilization of an appropriate gel filtration resin in a column format, for example, the relatively larger complex may be separated from the relatively smaller uncomplexed components. Similarly, the relatively different charge properties of the complex as compared to the uncomplexed molecules may be exploited to differentially separate the complex from the remaining individual reactants, for example through the use of ion-exchange chromatography resins. Such resins and chromatographic techniques are well known to one skilled in the art (see, e.g., Heegaard, 1998, J. Mol. Recognit. 11:141-148; Hage and Tweed, 1997, J. Chromatogr. B. Biomed. Sci. Appl., 699:499-525). Gel electrophoresis may also be employed to separate complexed molecules from unbound species (see, e.g., Ausubel et al (eds.), In: Current Protocols in Molecular Biology, J. Wiley & Sons, New York. 1999). In this technique, protein or nucleic acid complexes are separated based on size or charge, for example. In order to maintain the binding interaction during the electrophoretic process, nondenaturing gels in the absence of reducing agent are typically used, but conditions appropriate to the particular interactants will be well known to one skilled in the art. Immunoprecipitation is another common technique utilized for the isolation of a protein-protein complex from solution (see, e.g., Ausubel et al (eds.), In: Current Protocols in Molecular Biology, J. Wiley & Sons, New York. 1999). In this technique, all proteins binding to an antibody specific to one of the binding molecules are precipitated from solution by conjugating the antibody to a polymer bead that may be readily collected by centrifugation. The bound assay components are released from the beads (through a specific proteolysis event or other technique well known in the art which will not disturb the protein-protein interaction in the complex), and a second immunoprecipitation step is performed, this time utilizing antibodies specific for the correspondingly different interacting assay component. In this manner, only formed complexes should remain attached to the beads. Variations in complex formation in both the presence and the absence of a test compound can be compared, thus offering information about the ability of the compound to modulate interactions between the marker protein and its binding partner.

Also within the scope of the present invention are methods for direct detection of interactions between the marker protein and its natural binding partner and/or a test compound in a homogeneous or heterogeneous assay system without further sample manipulation. For example, the technique of fluorescence energy transfer may be utilized (see, e.g., Lakowicz et al, U.S. Pat. No. 5,631,169; Stavrianopoulos et al, U.S. Pat. No. 4,868,103). Generally, this technique involves the addition of a fluorophore label on a first ‘donor’ molecule (e.g., marker or test compound) such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, ‘acceptor’ molecule (e.g., marker or test compound), which in turn is able to fluoresce due to the absorbed energy. Alternately, the ‘donor’ protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the ‘acceptor’ molecule label may be differentiated from that of the ‘donor’. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, spatial relationships between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the ‘acceptor’ molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter). A test substance which either enhances or hinders participation of one of the species in the preformed complex will result in the generation of a signal variant to that of background. In this way, test substances that modulate interactions between a marker and its binding partner can be identified in controlled assays.

In another embodiment, modulators of marker expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of marker mRNA or protein in the cell, is determined. The level of expression of marker mRNA or protein in the presence of the candidate compound is compared to the level of expression of marker mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of marker expression based on this comparison. For example, when expression of marker mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of marker mRNA or protein expression. Conversely, when expression of marker mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of marker mRNA or protein expression. The level of marker mRNA or protein expression in the cells can be determined by methods described herein for detecting marker mRNA or protein.

In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell-based or a cell free assay, and the ability of the agent to modulate the activity of a marker protein can be further confirmed in vivo, e.g., in a whole animal model for cellular transformation and/or tumorigenesis.

This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a marker modulating agent, an antisense marker nucleic acid molecule, a marker-specific antibody, or a marker-binding partner) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

It is understood that appropriate doses of small molecule agents and protein or polypeptide agents depends upon a number of factors within the knowledge of the ordinarily skilled physician, veterinarian, or researcher. The dose(s) of these agents will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the agent to have upon the nucleic acid or polypeptide of the invention. Exemplary doses of a small molecule include milligram or microgram amounts per kilogram of subject or sample weight (e.g. about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram). Exemplary doses of a protein or polypeptide include gram, milligram or microgram amounts per kilogram of subject or sample weight (e.g. about 1 microgram per kilogram to about 5 grams per kilogram, about 100 micrograms per kilogram to about 500 milligrams per kilogram, or about 1 milligram per kilogram to about 50 milligrams per kilogram). It is furthermore understood that appropriate doses of one of these agents depend upon the potency of the agent with respect to the expression or activity to be modulated. Such appropriate doses can be determined using the assays described herein. When one or more of these agents is to be administered to an animal (e.g. a human) in order to modulate expression or activity of a polypeptide or nucleic acid of the invention, a physician, veterinarian, or researcher can, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific agent employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediamine-tetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For vintravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF; Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a polypeptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium, and then incorporating the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, acceptable methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches, and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes having monoclonal antibodies incorporated therein or thereon) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

For antibodies, the dosage may be 0.1 mg/kg to 100 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration. A method for lipidation of antibodies is described by Cruikshank et al. (1997) J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193.

The invention also provides vaccine compositions for the prevention and/or treatment of thymic lymphoma or hamartomatous tumors. The invention provides thymic lymphoma or hamartomatous tumor vaccine compositions in which a protein of a marker of Tables 1, 2, 3, or 4, or a combination of proteins of the markers of Tables 1, 2, 3, or 4, are introduced into a subject in order to stimulate an immune response against the thymic lymphoma or hamartomatous tumors. The invention also provides thymic lymphoma or hamartomatous tumor vaccine compositions in which a gene expression construct, which expresses a marker or fragment of a marker identified in Tables 1, 2, 3, or 4, is introduced into the subject such that a protein or fragment of a protein encoded by a marker of Tables 1, 2, 3, or 4 is produced by transfected cells in the subject at a higher than normal level and elicits an immune response.

In one embodiment, a thymic lymphoma or hamartomatous tumor vaccine is provided and employed as an immunotherapeutic agent for the prevention of thymic lymphoma or hamartomatous tumors. In another embodiment, a thymic lymphoma or hamartomatous tumor vaccine is provided and employed as an immunotherapeutic agent for the treatment of thymic lymphoma or hamartomatous tumors.

By way of example, a thymic lymphoma or hamartomatous tumor vaccine comprised of the proteins of the markers of Tables 1, 2, 3, or 4 may be employed for the prevention and/or treatment of thymic lymphoma or hamartomatous tumors in a subject by administering the vaccine by a variety of routes, e.g., intradermally, subcutaneously, or intramuscularly. In addition, the thymic lymphoma or hamartomatous tumor vaccine can be administered together with adjuvants and/or immunomodulators to boost the activity of the vaccine and the subject's response. In one embodiment, devices and/or compositions containing the vaccine, suitable for sustained or intermittent release could be, implanted in the body or topically applied thereto for the relatively slow release of such materials into the body. The thymic lymphoma or hamartomatous tumor vaccine can be introduced along with immunomodulatory compounds, which can alter the type of immune response produced in order to produce a response which will be more effective in eliminating the cancer.

In another embodiment, a thymic lymphoma or hamartomatous tumor vaccine comprised of an expression construct of the markers of Tables 1, 2, 3, or 4 may be introduced by injection into muscle or by coating onto microprojectiles and using a device designed for the purpose to fire the projectiles at high speed into the skin. The cells of the subject will then express the protein(s) or fragments of proteins of the markers of Tables 1, 2, 3, or 4 and induce an immune response. In addition, the thymic lymphoma or hamartomatous tumor vaccine may be introduced along with expression constructs for immunomodulatory molecules, such as cytokines, which may increase the immune response or modulate the type of immune response produced in order to produce a response which will be more effective in eliminating the cancer.

The marker nucleic acid molecules can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Pat. No. 5,328,470), or by stereotactic injection (see, e.g., Chen et aL, 1994, Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g. retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

V. Predictive Medicine

The present invention pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trails are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining the level of expression of one or more marker proteins or nucleic acids, in order to determine whether an individual is at risk of developing thymic lymphoma or hamartomatous tumors. Such assays can be used for prognostic or predictive purposes to thereby prophylactically treat an individual prior to the onset of the cancer.

Yet another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs or other compounds administered either to inhibit thymic lymphoma or hamartomatous tumors or to treat or prevent any other disorder {i.e. in order to understand any carcinogenic effects that such treatment may have}) on the expression or activity of a marker of the invention in clinical trials. These and other agents are described in further detail in the following sections.

A. Diagnostic Assays

An exemplary method for detecting the presence or absence of a marker protein or nucleic acid in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting the polypeptide or nucleic acid (e.g., mRNA, genomic DNA, or cDNA). The detection methods of the invention can thus be used to detect mRNA, protein, cDNA, or genomic DNA, for example, in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of a marker protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. In vitro techniques for detection of genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of a marker protein include introducing into a subject a labeled antibody directed against the protein or fragment thereof. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

A general principle of such diagnostic and prognostic assays involves preparing a sample or reaction mixture that may contain a marker, and a probe, under appropriate conditions and for a time sufficient to allow the marker and probe to interact and bind, thus forming a complex that can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways.

For example, one method to conduct such an assay would involve anchoring the marker or probe onto a solid phase support, also referred to as a substrate, and detecting target marker/probe complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, a sample from a subject, which is to be assayed for presence and/or concentration of marker, can be anchored onto a carrier or solid phase support. In another embodiment, the reverse situation is possible, in which the probe can be anchored to a solid phase and a sample from a subject can be allowed to react as an unanchored component of the assay.

There are many established methods for anchoring assay components to a solid phase. These include, without limitation, marker or probe molecules which are immobilized through conjugation of biotin and streptavidin. Such biotinylated assay components can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). In certain embodiments, the surfaces with immobilized assay components can be prepared in advance and stored.

Other suitable carriers or solid phase supports for such assays include any material capable of binding the class of molecule to which the marker or probe belongs. Well-known supports or carriers include, but are not limited to, glass, polystyrene, nylon, polypropylene, nylon, polyethylene, dextran, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.

In order to conduct assays with the above mentioned approaches, the non-immobilized component is added to the solid phase upon which the second component is anchored. After the reaction is complete, uncomplexed components may be removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized upon the solid phase. The detection of marker/probe complexes anchored to the solid phase can be accomplished in a number of methods outlined herein.

In one embodiment, the probe, when it is the unanchored assay component, can be labeled for the purpose of detection and readout of the assay, either directly or indirectly, with detectable labels discussed herein and which are well-known to one skilled in the art.

It is also possible to directly detect marker/probe complex formation without further manipulation or labeling of either component (marker or probe), for example by utilizing the technique of fluorescence energy transfer (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). A fluorophore label on the first, ‘donor’ molecule is selected such that, upon excitation with incident light of appropriate wavelength, its emitted fluorescent energy will be absorbed by a fluorescent label on a second ‘acceptor’ molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the ‘donor’ protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the ‘acceptor’ molecule label may be differentiated from that of the ‘donor’. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, spatial relationships between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the ‘acceptor’ molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).

In another embodiment, determination of the ability of a probe to recognize a marker can be accomplished without labeling either assay component (probe or marker) by utilizing a technology such as real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C., 1991, Anal Chem. 63:2338-2345 and Szabo et al., 1995, Curr. Opin. Struct. Biol. 5:699-705). As used herein, “BIA” or “surface plasmon resonance” is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.

Alternatively, in another embodiment, analogous diagnostic and prognostic assays can be conducted with marker and probe as solutes in a liquid phase. In such an assay, the complexed marker and probe are separated from uncomplexed components by any of a number of standard techniques, including but not limited to: differential centrifugation, chromatography, electrophoresis and immunoprecipitation. In differential centrifugation, marker/probe complexes may be separated from uncomplexed assay components through a series of centrifugal steps, due to the different sedimentation equilibria of complexes based on their different sizes and densities (see, for example, Rivas, G., and Minton, A. P., 1993, Trends Biochem Sci. 18(8):284-7). Standard chromatographic techniques may also be utilized to separate complexed molecules from uncomplexed ones. For example, gel filtration chromatography separates molecules based on size, and through the utilization of an appropriate gel filtration resin in a column format, for example, the relatively larger complex may be separated from the relatively smaller uncomplexed components. Similarly, the relatively different charge properties of the marker/probe complex as compared to the uncomplexed components may be exploited to differentiate the complex from uncomplexed components, for example through the utilization of ion-exchange chromatography resins. Such resins and chromatographic techniques are well known to one skilled in the art (see, e.g., Heegaard, N. H., 1998, J. Mol. Recognit. Winter 11(1-6):141-8; Hage, D. S., and Tweed, S. A. J Chromatogr B Biomed Sci Appl October 1997 10; 699(1-2):499-525). Gel electrophoresis may also be employed to separate complexed assay components from unbound components (see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987-1999). In this technique, protein or nucleic acid complexes are separated based on size or charge, for example. In order to maintain the binding interaction during the electrophoretic process, non-denaturing gel matrix materials and conditions in the absence of reducing agent are typically used. Appropriate conditions to the particular assay and components thereof will be well known to one skilled in the art.

In a particular embodiment, the level of marker mRNA can be determined both by in situ and by in vitro formats in a biological sample using methods known in the art. The term “biological sample” is intended to include tissues, cells, biological fluids and isolates thereof, isolated from a subject, as well as tissues, cells and fluids present within a subject. Many expression detection methods use isolated RNA. For in vitro methods, any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from thymic lymphoma or hamartomatous tumor cells, or from non-cancer cells (see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York 1987-1999). Additionally, large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski (1989, U.S. Pat. No. 4,843,155).

The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to a mRNA or genomic DNA encoding a marker of the present invention. Other suitable probes for use in the diagnostic assays of the invention are described herein. Hybridization of an mRNA with the probe indicates that the marker in question is being expressed.

In one format, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the markers of the present invention.

An alternative method for determining the level of mRNA marker in a sample involves the process of nucleic acid amplification, e.g., by rtPCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA, 88:189-193), self sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5′ or 3′ regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

For in situ methods, mRNA does not need to be isolated from the thymic lymphoma or hamartomatous tumor cells, or from non-cancer cells, prior to detection. In such methods, a cell or tissue sample is prepared/processed using known histological methods. The sample is then immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the marker.

As an alternative to making determinations based on the absolute expression level of the marker, determinations may be based on the normalized expression level of the marker. Expression levels are normalized by correcting the absolute expression level of a marker by comparing its expression to the expression of a gene that is not a marker, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene, or epithelial cell-specific genes. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, e.g., a non-cancerous sample, or between samples from different sources.

Alternatively, the expression level can be provided as a relative expression level.

To determine a relative expression level of a marker, the level of expression of the marker is determined for 10 or more samples of normal versus cancer cell isolates, preferably 50 or more samples, prior to the determination of the expression level for the sample in question. The mean expression level of each of the genes assayed in the larger number of samples is determined and this is used as a baseline expression level for the marker. The expression level of the marker determined for the test sample (absolute level of expression) is then divided by the mean expression value obtained for that marker. This provides a relative expression level.

Preferably, the samples used in the baseline determination will be from thymic lymphoma or hamartomatous tumor cells, or from non-cancerous cells of the same tissue type. The choice of the cell source is dependent on the use of the relative expression level. Using expression found in normal tissues as a mean expression score aids in validating whether the marker assayed is tissue-specific (versus cells unaffected by thymic lymphoma or hamartomatous tumors). In addition, as more data is accumulated, the mean expression value can be revised, providing improved relative expression values based on accumulated data. Expression data from thymic lymphoma and hamartomatous tumor cells, and from non-cancerous cells, provides a means for grading the severity of the thymic lymphoma or hamartomatous tumor state.

In another embodiment of the present invention, a marker protein is detected. An agent for detecting marker proteins of the invention may be an antibody capable of binding to such a protein or a fragment thereof, preferably an antibody with a detectable label. Antibodies can be monoclonal but may also be polyclonal. An intact antibody, or a fragment or derivative thereof (e.g., Fab or F(ab′)₂) can be used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.

Cellular proteins can be isolated using techniques that are well known to those of skill in the art. The protein isolation methods employed can, for example, be such as those described in Harlow and Lane (Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

A variety of formats can be employed to determine whether a sample contains a protein that binds to a given antibody. Examples of such formats include, but are not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis and enzyme linked immunoabsorbant assay (ELISA). A skilled artisan can readily adapt known protein/antibody detection methods for use in determining whether thymic lymphoma or hamartomatous tumor cells express a marker of the present invention.

In one format, antibodies, or antibody fragments or derivatives, can be used in methods such as Western blots or immunofluorescence techniques to detect the expressed proteins. In such uses, it is generally preferable to immobilize either the antibody or proteins on a solid support. Suitable solid phase supports or carriers include any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.

One skilled in the art will know many other suitable carriers for binding antibody or antigen, and will be able to adapt such support for use with the present invention. For example, protein isolated from thymic lymphoma or hamartomatous tumor cells can be run on a polyacrylamide gel electrophoresis and immobilized onto a solid phase support such as nitrocellulose. The support can then be washed with suitable buffers followed by treatment with the detectably labeled antibody. The solid phase support can then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on the solid support can then be detected by conventional means.

The invention also encompasses kits for detecting the presence of a marker protein or nucleic acid in a biological sample (e.g., tissue, body fluid). Such kits can be used to determine if a subject is suffering from or is at increased risk of developing thymic lymphoma or hamartomatous tumors. For example, the kit can comprise a labeled compound or agent capable of detecting a marker protein or nucleic acid in a biological sample and means for determining the amount of the protein or mRNA in the sample (e.g., an antibody which binds the protein or a fragment thereof, or an oligonucleotide probe which binds to DNA or mRNA encoding the protein). Kits can also include instructions for interpreting the results obtained using the kit.

For antibody-based kits, the kit can comprise, for example: (1) a first antibody (e.g., attached to a solid support) which binds to a marker protein; and, optionally, (2) a second, different antibody which binds to either the protein or the first antibody and is conjugated to a detectable label.

For oligonucleotide-based kits, the kit can comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a marker protein or (2) a pair of primers useful for amplifying a marker nucleic acid molecule. The kit can also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent. The kit can further comprise components necessary for detecting the detectable label (e.g., an enzyme or a substrate). The kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.

B. Pharmacogenomics

The markers of the invention are also useful as pharmacogenomic markers. As used herein, a “pharmacogenomic marker” is an objective biochemical marker whose expression level correlates with a specific clinical drug response or susceptibility in a patient (see, e.g., McLeod et al. (1999) Eur. J. Cancer 35(12): 1650-1652). The presence or quantity of the pharmacogenomic marker expression is related to the predicted response of the patient and more particularly the patient's tumor to therapy with a specific drug or class of drugs. By assessing the presence or quantity of the expression of one or more pharmacogenomic markers in a patient, a drug therapy which is most appropriate for the patient, or which is predicted to have a greater degree of success, may be selected. For example, based on the presence or quantity of RNA or protein encoded by specific tumor markers in a patient, a drug or course of treatment may be selected that is optimized for the treatment of the specific tumor likely to be present in the patient. The use of pharmacogenomic markers therefore permits selecting or designing the most appropriate treatment for each cancer patient without trying different drugs or regimes.

Another aspect of pharmacogenomics deals with genetic conditions that alter the way the body acts on drugs. These pharmacogenetic conditions can occur either as rare defects or as polymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, a PM will show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.

Thus, the level of expression of a marker of the invention in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual. In addition, pharmacogenetic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a modulator of expression of a marker of the invention.

C. Monitoring Clinical Trials

Monitoring the influence of agents (e.g., drug compounds) on the level of expression of a marker of the invention can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent to affect marker expression can be monitored in clinical trials of subjects receiving treatment for thymic lymphoma or hamartomatous tumors. In one embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of one or more selected markers of the invention in the pre-administration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression of the marker(s) in the post-administration samples; (v) comparing the level of expression of the marker(s) in the pre-administration sample with the level of expression of the marker(s) in the post-administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, for the markers provided in Tables 1 and 3, increased expression of the marker gene(s) during the course of treatment may indicate ineffective dosage and the desirability of increasing the dosage. Conversely, decreased expression of the marker gene(s) may indicate efficacious treatment and no need to change dosage. Likewise, for the markers provided in Tables 2 and 4, decreased expression of the marker gene(s) during the course of treatment may indicate ineffective dosage and the desirability of increasing the dosage. Conversely, increased expression of the marker gene(s) may indicate efficacious treatment and no need to change dosage.

D. Electronic Apparatus Readable Media and Arrays

Electronic apparatus readable media comprising a marker of the present invention is also provided. As used herein, “electronic apparatus readable media” refers to any suitable medium for storing, holding or containing data or information that can be read and accessed directly by an electronic apparatus. Such media can include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as compact disc; electronic storage media such as RAM, ROM, EPROM, EEPROM and the like; general hard disks and hybrids of these categories such as magnetic/optical storage media. The medium is adapted or configured for having recorded thereon a marker of the present invention.

As used herein, the term “electronic apparatus” is intended to include any suitable computing or processing apparatus or other device configured or adapted for storing data or information. Examples of electronic apparatus suitable for use with the present invention include stand-alone computing apparatus; networks, including a local area network (LAN), a wide area network (WAN) Internet, Intranet, and Extranet; electronic appliances such as a personal digital assistants (PDAs), cellular phone, pager and the like; and local and distributed processing systems.

As used herein, “recorded” refers to a process for storing or encoding information on the electronic apparatus readable medium. Those skilled in the art can readily adopt any of the presently known methods for recording information on known media to generate manufactures comprising the markers of the present invention.

A variety of software programs and formats can be used to store the marker information of the present invention on the electronic apparatus readable medium. For example, the marker nucleic acid sequence can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and MicroSoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like, as well as in other forms. Any number of data processor structuring formats (e.g., text file or database) may be employed in order to obtain or create a medium having recorded thereon the markers of the present invention.

By providing the markers of the invention in readable form, one can routinely access the marker sequence information for a variety of purposes. For example, one skilled in the art can use the nucleotide or amino acid sequences of the present invention in readable form to compare a target sequence or target structural motif with the sequence information stored within the data storage means. Search means are used to identify fragments or regions of the sequences of the invention which match a particular target sequence or target motif.

The present invention therefore provides a medium for holding instructions for performing a method for determining whether a subject has thymic lymphoma or hamartomatous tumors, or a pre-disposition to the development of thymic lymphoma or hamartomatous tumors, wherein the method comprises the steps of determining the presence or absence of a marker and based on the presence or absence of the marker, determining whether the subject has thymic lymphoma or hamartomatous tumors, or a pre-disposition to the development of thymic lymphoma or hamartomatous tumors, and/or recommending a particular treatment for thymic lymphoma or hamartomatous tumors or a pre-cancerous condition.

The present invention further provides in an electronic system and/or in a network, a method for determining whether a subject has thymic lymphoma or hamartomatous tumors, or a pre-disposition to thymic lymphoma or hamartomatous tumors associated with a marker wherein the method comprises the steps of determining the presence or absence of the marker, and based on the presence or absence of the marker, determining whether the subject has thymic lymphoma or hamartomatous tumors, or a pre-disposition to the development of thymic lymphoma or hamartomatous tumors, and/or recommending a particular treatment for the thymic lymphoma or hamartomatous tumor or pre-cancerous condition. The method may further comprise the step of receiving phenotypic information associated with the subject and/or acquiring from a network phenotypic information associated with the subject.

The present invention also provides in a network, a method for determining whether a subject has thymic lymphoma or hamartomatous tumors, or a pre-disposition to thymic lymphoma or hamartomatous tumors associated with a marker, said method comprising the steps of receiving information associated with the marker, receiving phenotypic information associated with the subject, acquiring information from the network corresponding to the marker and/or thymic lymphoma or hamartomatous tumor, and based on one or more of the phenotypic information, the marker, and the acquired information, determining whether the subject has a thymic lymphoma or hamartomatous tumor, or a pre-disposition to the development of thymic lymphoma or hamartomatous tumors. The method may further comprise the step of recommending a particular treatment for the thymic lymphoma or hamartomatous tumor or pre-cancerous condition.

The present invention also provides a business method for determining whether a subject has thymic lymphoma or hamartomatous tumors, or a pre-disposition to the development of thymic lymphoma or hamartomatous tumors, said method comprising the steps of receiving information associated with the marker, receiving phenotypic information associated with the subject, acquiring information from the network corresponding to the marker and/or thymic lymphoma or hamartomatous tumor, and based on one or more of the phenotypic information, the marker, and the acquired information, determining whether the subject has thymic lymphoma or hamartomatous tumors, or a pre-disposition to the development of thymic lymphoma or hamartomatous tumors. The method may further comprise the step of recommending a particular treatment for the thymic lymphoma or hamartomatous tumor or pre-cancer condition.

The invention also includes an array comprising a marker of the present invention. The array can be used to assay expression of one or more genes in the array. In one embodiment, the array can be used to assay gene expression in a tissue to ascertain tissue specificity of genes in the array. In this manner, up to about 7600 genes can be simultaneously assayed for expression. This allows a profile to be developed showing a battery of genes specifically expressed in one or more tissues.

In addition to such qualitative determination, the invention allows the quantitation of gene expression. Thus, not only tissue specificity, but also the level of expression of a battery of genes in the tissue is ascertainable. Thus, genes can be grouped on the basis of their tissue expression per se and level of expression in that tissue. This is useful, for example, in ascertaining the relationship of gene expression between or among tissues. Thus, one tissue can be perturbed and the effect on gene expression in a second tissue can be determined. In this context, the effect of one cell type on another cell type in response to a biological stimulus can be determined. Such a determination is useful, for example, to know the effect of cell-cell interaction at the level of gene expression. If an agent is administered therapeutically to treat one cell type but has an undesirable effect on another cell type, the invention provides an assay to determine the molecular basis of the undesirable effect and thus provides the opportunity to co-administer a counteracting agent or otherwise treat the undesired effect. Similarly, even within a single cell type, undesirable biological effects can be determined at the molecular level. Thus, the effects of an agent on expression of other than the target gene can be ascertained and counteracted.

In another embodiment, the array can be used to monitor the time course of expression of one or more genes in the array. This can occur in various biological contexts, as disclosed herein, for example development of thymic lymphoma or hamartomatous tumors, progression of the thymic lymphoma or hamartomatous tumor, and processes, such a cellular transformation associated with thymic lymphoma or hamartomatous tumors.

The array is also useful for ascertaining the effect of the expression of a gene on the expression of other genes in the same cell or in different cells. This provides, for example, for a selection of alternate molecular targets for therapeutic intervention if the ultimate or downstream target cannot be regulated.

The array is also useful for ascertaining differential expression patterns of one or more genes in normal and abnormal cells. This provides a battery of genes that could serve as a molecular target for diagnosis or therapeutic intervention.

E. Surrogate Markers

The markers of the invention may serve as surrogate markers for one or more disorders or disease states or for conditions leading up to disease states, and in particular, thymic lymphoma or hamartomatous tumors. As used herein, a “surrogate marker” is an objective biochemical marker which correlates with the absence or presence of a disease or disorder, or with the progression of a disease or disorder (e.g., with the presence or absence of a tumor). The presence or quantity of such markers is independent of the disease. Therefore, these markers may serve to indicate whether a particular course of treatment is effective in lessening a disease state or disorder. Surrogate markers are of particular use when the presence or extent of a disease state or disorder is difficult to assess through standard methodologies (e.g., early stage tumors), or when an assessment of disease progression is desired before a potentially dangerous clinical endpoint is reached (e.g., an assessment of cardiovascular disease may be made using cholesterol levels as a surrogate marker, and an analysis of HIV infection may be made using HIV RNA levels as a surrogate marker, well in advance of the undesirable clinical outcomes of myocardial infarction or fully-developed AIDS). Examples of the use of surrogate markers in the art include: Koomen et al. (2000) J. Mass. Spectrom. 35: 258-264; and James (1994) AIDS Treatment News Archive 209.

The markers of the invention are also useful as pharmacodynamic markers. As used herein, a “pharmacodynamic marker” is an objective biochemical marker which correlates specifically with drug effects. The presence or quantity of a pharmacodynamic marker is not related to the disease state or disorder for which the drug is being administered; therefore, the presence or quantity of the marker is indicative of the presence or activity of the drug in a subject. For example, a pharmacodynamic marker may be indicative of the concentration of the drug in a biological tissue, in that the marker is either expressed or transcribed or not expressed or transcribed in that tissue in relationship to the level of the drug. In this fashion, the distribution or uptake of the drug may be monitored by the pharmacodynamic marker. Similarly, the presence or quantity of the pharmacodynamic marker may be related to the presence or quantity of the metabolic product of a drug, such that the presence or quantity of the marker is indicative of the relative breakdown rate of the drug in vivo. Pharmacodynamic markers are of particular use in increasing the sensitivity of detection of drug effects, particularly when the drug is administered in low doses. Since even a small amount of a drug may be sufficient to activate multiple rounds of marker transcription or expression, the amplified marker may be in a quantity which is more readily detectable than the drug itself. Also, the marker may be more easily detected due to the nature of the marker itself; for example, using the methods described herein, antibodies may be employed in an immune-based detection system for a protein marker, or marker-specific radiolabeled probes may be used to detect a mRNA marker. Furthermore, the use of a pharmacodynamic marker may offer mechanism-based prediction of risk due to drug treatment beyond the range of possible direct observations. Examples of the use of pharmacodynamic markers in the art include: Matsuda et al. U.S. Pat. No. 6,033,862; Hattis et aL (1991) Env. Health Perspect. 90: 229-238; Schentag (1999) Am. J Health-Syst. Pharm. 56 Suppl. 3: S21-S24; and Nicolau (1999) Am, J. Health-Syst. Pharm. 56 Suppl. 3: S16-S20.

The subject matter described herein may be better understood by reference to the following examples, which are provided by way of exemplification and are in no way limiting.

Example 1

Somatic Deletion of all Three FoxO Genes Results in Lineage-Restricted Tumor Phenotypes.

To genetically assess the cancer relevance of the FoxO members in vivo, null and conditional alleles for all three FoxO genes were generated (Example 2 and FIGS. 6, 7). As detailed in Example 2 (FIGS. 8-11, Table 5), exhaustive characterization of mice bearing single and compound germline FoxO null mutations, including mice retaining only a single functional FoxO1 allele (FoxO1^(−/+); FoxO3^(−/−); FoxO4^(−/−)) revealed remarkably mild cancer predisposition phenotypes as a function of advancing age or following carcinogen treatment. The potential for redundancy and developmental compensation among the FoxOs, coupled with the embryonic lethality of FoxO1 deficiency (Furuyama et al., 2004; Hosaka et al., 2004), prompted the use of the Mx-Cre transgene to achieve widespread somatic FoxO deletion in adult tissues. The Mx1 promoter is activated by interferon or double-stranded RNA (polyinosine-polycytidylic acid (pI-pC)), and pI-pC injection at 4-5 weeks of age results in transient widespread Cre expression, particularly robust in hematopoietic and liver compartments, and to a moderate extent across many other tissues including epithelial cells (Kuhn et al., 1995) (see Mx-directed tissue distribution of Cre below). By generating mice harboring this Mx-Cre transgene and various combinations of the conditional FoxO1^(L), FoxO3^(L), and FoxO4^(L) alleles, an in vivo system was established in which to examine the biological consequences of FoxO family deletion in specific tissues of the adult mouse.

To delete the FoxO genes in vivo, Mx-Cre⁺;FoxO1/3/4^(L/L) animals were injected with pI-pC. For controls, littermate Mx-Cre⁻; FoxO1/3/4^(L/L) mice were similarly treated with pI-pC (hereafter designated as “Mx-Cre⁺” and “Mx-Cre⁻ ”, respectively). By 19-30 weeks of age, a significant number of the Mx-Cre⁺ mice developed aggressive CD4⁺ CD8⁺ lymphoblastic thymic lymphomas filling the chest cavity, with spread to spleen, liver, and lymph nodes (FIG. 1a, b, and f , p<0.0001). While Mx-Cre-mediated gene deletion in the thymus was incomplete (Kuhn et al., 1995) (FIG. 1c ), the resultant lymphomas display an enrichment of the null alleles for all three FoxO genes (FIG. 1c ) accompanied by a marked decrease of FoxO expression on both mRNA and protein levels (FIGS. 1d and e ). This molecular profile and the lack of lymphomas in all other genotypes retaining at least one FoxO allele (FIG. 1a ), is consistent with the need for complete FoxO deficiency in lymphomagenesis.

In addition, all Mx-Cre⁺ mice developed a striking age-progressive hamartomatous phenotype in the endothelial cell lineage, with numerous hemangiomas in different tissues, resulting in premature death (FIG. 2a , p<0.0001; overall median survival of 42.6 weeks or 51.3 weeks for lymphoma-free mice). Hemangiomas were most exaggerated in the uterus—first evident by 6-8 weeks and progressing to distorted tissue consisting almost entirely of large blood-filled spaces by 30-40 weeks (FIG. 2b ). Histological examination of the uterine lesions revealed massive hemangiomas with intraluminal blood and numerous thrombi (FIG. 2c ). Hemangiomas were also observed in other anatomic locations including skeletal muscle, abdominal wall, liver, adrenal glands, bone marrow, omentum, lymph node, and skin (FIG. 2c ; FIG. 12a ). The endothelial cells in most lesions were histologically benign; however, 9% of Mx-Cre⁺ mice progressed to lethal malignant angiosarcomas (FIG. 2c ).

To further investigate this vascular phenotype, endothelial cells were labelled with fluorescein-conjugated lectin, which readily revealed significant increases in the number of endothelial cells in affected organs (FIG. 2d ). This proliferative phenotype was apparent as early as three weeks after pI-pC injection (i.e., 7 weeks in age), prior to onset of any macroscopic abnormalities in these vascular beds. In contrast, endothelial cells in the lung and kidney (where hemangiomas do not develop following documented FoxO deletion) did not show any abnormalities even at 27 weeks after pI-pC injection (i.e., 31 weeks in age), clearly establishing tissue-context requirements for the FoxOs in vivo across this single lineage (FIG. 2d ; data not shown).

Further support for a role of FoxOs in this vascular phenotype stems from observations of similar but less severe hemangiomas in Mx-Cre⁺ mice with deficiency of only one or two FoxO genes. Here, by 60 weeks of age, 100% of Mx-Cre⁺; FoxO1^(L/L) females displayed mild hemangiomatous anomalies in the uterus and occasionally in perirenal fat, with no abnormalities in other tissues (FIG. 12b ). Mx-Cre⁺;FoxO1/4^(L/L) mice displayed mild hemangiomatous changes in the uterus and occasionally in other tissues (FIG. 12c ), and all Mx-Cre⁺;FoxO1/3^(L/L) mice displayed vascular abnormalities with similar tissue distribution as Mx-Cre⁺;FoxO1/3/4^(L/L) mice (FIG. 12d ). Importantly, these hemangiomatous lesions in the FoxO1 deficient mice retaining either FoxO3 or FoxO4 activity were significantly less severe as they did not lead to mortality prior to 50 weeks of age (FIG. 2a ). In contrast, vascular abnormalities were not detected in Mx-Cre⁺;FoxO3^(L/L) mice up to 21 weeks and in germline FoxO3^(−/−); FoxO4⁻¹ mice up to 2 years of age and FoxO1^(−/+); FoxO3^(−/−); FoxO4^(−/−) mice up to 40 weeks of age. These genotype-phenotype correlations point to FoxO1 as the most potent regulator of adult vascular homeostasis, with lesser but discernable contributions from the other FoxOs. Vascular lesions were much more pervasive and severe following elimination of all three FoxOs, and premature mortality attributable to these vascular lesions was observed only in mice with somatic deletion of all three FoxOs (FIG. 2a ).

Cell Biological Impact of FoxO Family Deletion.

As a first step in understanding how the FoxO family constrains development of thymic lymphoma and hemangiomas, the proliferative and survival profiles of thymocyte and endothelial cells derived from young cancer-free mice of various genotypes were examined. Although histologically normal (data not shown) albeit with a slight reduction in total number of thymocytes compared to Mx-Cre⁻ controls (99,110,000±13,590,000 versus 147,300,000±20,950,000; n=6 each; p=0.0825), the Mx-Cre⁺ thymocytes exhibited altered responses to growth and death stimuli. Upon stimulation with PMA+ionomycin, Mx-Cre⁺ thymocytes showed increased proliferation relative to Mx-Cre⁻ controls (89.5±19.5 vs 38.2±1.6 fold, p=0.058, FIG. 3a ). Proliferative enhancement was also observed with CD3+IL2 stimulation (5.4±1.5 vs 1.7±0.3 fold, p=0.07, FIG. 3a ). Conversely, Mx-Cre⁺ thymocytes were more resistant to cell death stimuli such as γ-irradiation and high-dose dexamethasome treatment (48.4±0.8 vs 22.2±2.3% survival, p=0.00075 and 69.3±0.7 vs 50.6±2.6% survival, p=0.0077 respectively, FIG. 3a ). These observations indicate that complete loss of FoxO gene function in thymocytes predisposes to lymphomagenesis through cell autonomous mechanisms that enhance cellular proliferation and survival.

Taking advantage of efficient Mx-Cre mediated recombination in endothelial cells in all tissue compartments examined (FIG. 13) (Hayashi et al., 2004; Schneider et al., 2003) and the organ-restricted hemangioma phenotype in Mx-Cre⁺ mice, the biological consequences of complete FoxO deletion in endothelial cells were compared from affected and unaffected tissues. To this end, endothelial cells (ECs) were isolated and cultured from the lung (which displayed no phenotype in vivo) and liver (which displayed prominent hemangiomatous changes) from age-matched pairs of mice. Consistent with the LacZ reporter mouse, Western blot analysis documented the absence of FoxO protein expression in both lung and liver ECs following activation of Mx-Cre with pI-pC (data not shown). Relative to Mx-Cre⁺ lung ECs, Mx-Cre⁺ liver ECs showed enhanced proliferative response to stimulation by VEGF and bFGF, two potent pro-angiogenic cytokines (Bikfalvi et al., 1997; Ferrara et al., 2003). As shown in FIG. 3b , while Mx-Cre⁻ and Mx-Cre⁺ lung ECs responded to VEGF similarly regardless of their FoxO status (41.7% vs. 47.2% increase in viability, p=0.332), Mx-Cre⁺ liver ECs exhibited a dramatic enhancement in their ability to proliferate upon VEGF stimulation relative to their FoxO-intact Mx-Cre⁻ counterpart (73.3% vs 17.9%, p=0.003). A similar trend was observed with bFGF stimulation (p=0.019; data not shown). Conversely, Mx-Cre⁺ liver ECs were significantly less sensitive to TGF-β1, a cytokine known to induce EC cytostasis through Smad signalling (Goumans et al., 2003). Here, Mx-Cre⁻ liver ECs showed reduced survival (˜60%) upon exposure to TGF-β1, whereas Mx-Cre⁺ liver ECs were much less sensitive (˜90%, p=0.03; FIG. 3b ). Lung ECs were insensitive to TGF-β1 stimulation irrespective of FoxO status (FIG. 3b ). Mirroring the in vivo phenotypes, FoxO-deficient liver ECs exhibited enhanced proliferative and survival potential, while FoxO-deficient lung ECs did not show detectable phenotypic alterations. That FoxO exerts these biological effects in a cell autonomous manner is supported by the observation of a prominent hemangiomatous phenotype in skeletal muscle, which showed a lack of Mx-Cre-mediated recombination in the surrounding skeletal muscle fibers yet efficient recombination in the endothelium (FIG. 13k ).

Transcriptome and Promoter Analysis Revealed Lineage-Restricted FoxO Activities In Vivo.

The above in vivo and in vitro phenotypic characterization shows that FoxO family functions in normal tissue homeostasis and in cancer suppression are not only lineage-restricted, but also organ-specific. To gain additional insights into the mechanistic basis for such specificity, a comparative transcription profiling analysis of purified lung and liver ECs following pI-pC treatment of age-matched Mx-Cre⁺ and Mx-Cre⁻ mice was conducted. It was hypothesized that normalization against phenotypically unaffected lung ECs would provide an effective biological filter for identifying physiologically relevant FoxO targets versus secondary/bystander transcriptional events—the latter would include genes whose expression respond to FoxO regulation but do not play a role in the observed phenotypes as well as those whose expression might be altered by activation of Cre expression during FoxO deletion. Here, transcriptome profiles of liver ECs with and without functional FoxOs were compared with those of lung ECs (see the Methods section below) to generate a list of 138 significantly differentially expressed genes—89 of which were upregulated and 49 downregulated in liver ECs, but not in lung ECs, upon documented FoxO deletion (Table 7). Consistent with the observed in vivo phenotypes, several differentially expressed genes have validated roles in endothelial cell biology, angiogenesis and tissue morphogenesis, such as XLKD1 (LYVE-1), VCAM1, Angiopoietin-like 4 (ANGPTL4), adrenomedullin (ADM), thromospondin1 (THBS1), and ID1, and extracellular matrix proteins such as fibrillin (FBN1) with well-established roles in the maintenance of vascular integrity (Benezra, 2001; Cook-Mills, 2002; Kato et al., 2005; Le Jan et al., 2003; Mouta Carreira et al., 2001; Ramirez et al., 1993).

It is well established that transcriptome profiles capture not only genes that are directly and causally altered in a particular state (such as FoxO deficiency), but also secondary and tertiary targets. It was reasoned that the identification of FoxO binding elements (BEs) in differentially expressed genes would provide more direct insight into FoxO's actions and yield targets with causal roles in the observed phenotypes. To that end, a systematic in silico analysis of the regulatory regions of these 138 genes was conducted. In particular, evolutionarily conserved FoxO consensus Bes were sought. For each gene, the −8 kb to +2 kb region surrounding the transcription start site and the 0 to +5 kb region downstream of the transcription end site in the mouse genome were surveyed. A position specific weight matrix (PWM) was constructed based on evolutionary conservation of canonical insulin-regulated FoxO targets (IGFBP1, G6PD, PEPCK) to characterize the FoxO binding motif (consensus=BBTRTTTTD) (D.H.C., unpublished data). Potential FoxO BEs were filtered further by cross-species conservation with two independent methodologies. In the first method, the “conservation score” for each BE by alignment of the mouse genome with 9 other vertebrate genomes was calculated, with higher scores indicating greater conservation. In the second method, cross-species alignments of each putative direct target with its orthologs in human, dog, cow, chicken and zebrafish were constructed and the FoxO matrix was used to scan each to determine whether that particular FoxO BE could be identified. BEs that could be identified in mouse, human and at least one other species were designated as a “3-species conserved” BE. By requiring at least one evolutionary conserved FoxO BEs predicted by both methods, 22 putative direct targets of FoxO family were identified in liver ECs, 9 of which were down-modulated and 13 up-modulated upon FoxO deletion. Several of these genes are highly relevant to cancer (e.g., MEIS1/PBX1, TCF4, and KLF6), vascular biology (e.g., CTGF), or both, such as ID1 and adrenomedullin, two factors known to play critical roles in endothelial cell survival and promote angiogenesis during development and tumorigenesis (Benezra, 2001; Kato et al., 2005; Nikitenko et al., 2006) (Tables 3 and 4).

To address whether downstream effectors of FoxOs are lineage-specific, a similar in silico promoter analysis was performed on a list of 354 most differentially expressed genes in the thymocyte lineage. It was found that putative direct targets with predicted conserved FoxO BEs and transcriptionally regulated by FoxOs in thymocytes were completely non-overlapping with those in EC lineage (Tables 1 and 2). A notable FoxO target in thymocytes is p27^(KIP1) (CDKN1B), one of the downregulated genes with the highest conservation score and number of three-species BE's. p27^(KIP1) is a well-documented FoxO target (Dijkers et al., 2000b; Medema et al., 2000; Stahl et al., 2002) and is also known to serve a major tumor suppressor role in T-cell lymphomas (Geisen et al., 2003; Kang-Decker et al., 2004; Martins and Berns, 2002). Misregulation of this known FoxO target is likely to contribute to the increased incidence of T-cell lymphomas following FoxO inactivation. This in silico analysis not only identified putative targets of direct FoxO regulation, but demonstrated in molecular terms that signalling downstream of FoxO is tissue- and cell-lineage specific, providing in part a molecular basis for the more restricted phenotypic impact of FoxO deletion relative to activated PI3K-AKT signalling.

Sprouty2 is a Downstream Effector of FoxO-Regulated Endothelial Cell Homeostasis

To more deeply elucidate the mechanistic basis of FoxO dysregulation in hemangioma development, Sprouty2 was further examined, as it represented the most significantly regulated gene with highest number of conserved FoxO BEs in affected liver EC but not in lung EC. First, chromatin immunoprecipitation analysis (ChIP) was performed to verify that Sprouty2 is a direct transcriptional target of FoxO. Using mixture of anti-FoxO1/3/4 antibodies, DNA fragments spanning FoxO BEs from both the proximal and distal regions of Sprouty2 gene were more efficiently co-immunoprecipitated in the FoxO-expressing liver ECs, versus those deficient for the FoxOs (FIG. 4b ). This result demonstrates that the FoxOs bind directly to their cognate BEs in vivo, thus validating the computational approaches employed in the identification of these putative direct targets.

Next, it was further documented, by immunoblotting and quantitative RT-PCR, that Sprouty2 was significantly and reproducibly downregulated in independently derived MxCre⁺ liver ECs, but not in MxCre⁺ lung ECs (FIG. 4c, d ). In addition, as an independent validation in another affected vascular bed, it was documented that Sprouty2 protein was readily detectable in the capillary endothelium in normal skeletal muscle but undetectable in Mx-Cre⁺ mice 3 weeks after treatment with pI-pC (FIG. 14). These in vivo results confirm that downregulation of Sprouty2 in the FoxO deficient vasculature precedes the development of vascular anomalies in specific, susceptible compartments.

Next, to determine the functional relevance of Sprouty2 in endothelium, cell biological consequences of Sprouty2 knockdown in Mx-Cre⁻ liver ECs were compared with those of Mx-Cre⁺ liver ECs. Using two independent Sprouty2 shRNAs (shSPRY2-1 and shSPRY2-2), FoxO protein knockdown of 50% to 80% was documented in primary cultures of liver ECs (FIG. 5c ). As shown in FIG. 5a , passage-associated proliferative arrest of Mx-Cre⁻ primary liver ECs could be reversed by Sprouty2 knockdown, conferring enhanced replicative potential to Mx-Cre⁻ ECs similar to that of FoxO-deficient Mx-Cre⁺ ECs. Correspondingly, increased cell cycle progression and decreased apoptosis could be demonstrated upon Sprouty2 knockdown. Specifically, the BrdU positive population in Mx-Cre⁻ liver EC cultures was increased from 6% to 13% and 9% with shSPRY2-1 and shSPRY2-2, respectively, comparable to the level of proliferation in Mx-Cre⁺ liver ECs (15%). Importantly, further reduction in Sprouty2 expression in Mx-Cre⁺ liver ECs had no additional effects (FIG. 5b ). Similarly, TUNEL positive nuclei in Mx-Cre⁻ liver ECs decreased from 19% to 7% (p<0.01), comparable to the 5% observed in Mx-Cre⁺ liver ECs (p<0.01). Associated with these phenotypic changes were differential expression of CyclinD1, p15, and p21 expression as well as Bim following Sprouty2 shRNA knockdown (FIG. 5d ), reinforcing the view that Sprouty2 functions as a negative regulator of endothelial cell proliferation and survival. Finally, in matrigel morphogenesis assays, Mx-Cre⁺ liver ECs developed a more robust tube network compared to Mx-Cre⁻ liver ECs (4123±318 vs 3229±285 μM, p=0.0057, FIG. 5e ). Again, knockdown of Sprouty2 in Mx-Cre⁻ liver ECs enhanced such VEGF-induced tube formation to levels approaching those of Mx-Cre⁺ liver ECs (3973±221 vs 3229±285 μm, p=0.0061, FIG. 5e ). It is possible that lack of complete phenotypic equivalence of Sprouty 2 knockdown and FoxO deficiency may point to contributions from other FoxO targets, but these findings demonstrate that Sprouty2 has essential roles in EC growth and tube morphogenesis and is a major effector of FoxO function in the endothelium.

Methods

Immunohistochemistry and Immunofluorescence.

Formalin-fixed, paraffin-embedded sections were used. For stains of pituitary tumors, sections were incubated with rabbit polyclonal antibodies to prolactin, GH, or ACTH (all obtained through NHPP, NIDDK, and Dr. A. F. Parlow) followed by incubation with HRP-goat anti-rabbit IgG(H+L) (Zymed) or with mouse monoclonal antibodies to TSH, FSH, and LH (Lab Vision/Neomarkers) utilizing the Histomouse-MAX kit (Zymed). In all cases, sections were then incubated with DAB (Vector) and then counterstained with hematoxylin. To visualize functional vasculature, deeply anesthetised mice were intravenously injected with 100 μg FITC-labeled tomato lectin (Vector Laboratories) and perfused the hearts with 4% paraformaldehyde; tissues were then frozen in OCT medium. For the detection of Sprouty2 in tissue sections, 5 micron frozen sections were blocked with 10% milk and incubated with rabbit anti-Sprouty2 antibody (1:100, Upstate Biotechnology) and secondarily detected using a Cy3-conjugated anti-rabbit secondary (1:500) mixed with MadCam antibody (MECA-FITC, 1:100, eBioscience). Images were acquired at a 0.5 micron thickness interval using a Zeiss inverted microscope (Axiovert) with automated stage, and image deconvolution was carried out with the nearest neighbor algorithm.

Thymocyte Isolation, Proliferation, and Apoptosis.

For analysis of thymocytes, fresh thymus was passed through a cell strainer (Becton Dickinson) in order to produce single cell suspensions for analysis and resuspended in RPMI-1640 media containing 10% fetal calf serum. For thymocyte proliferation assays, cells were seeded in 96-well plates at 1×10⁵ cells per well and subjected to the following conditions: no mitogen, PMA (1 ng/ml)+ionomycin (50 ng/ml), and 3 μg/ml anti-CD3e (eBioscience)+100 U IL-2. Cells were grown for 48 hours, pulsed with 1 μCi [3H]-thymidine (Amersham) for the last 16 hours. Incorporated [3H]-thymidine was measured using the 1450 MicroBeta Trilux Scintillation and Luminescence Counter. For analysis of apoptosis, thymocytes were plated in 12-well plates (1×10⁶ cells per well) and treated with 5 Gy gamma-irradiation or 1 μM dexamethasone for 18 hours. Cell viability was determined by staining the cells with propidium iodide and performing flow cytometric analysis.

Flow Cytometric Analysis.

Single cell suspensions of thymus were incubated with the following monoclonal antibodies: anti-CD4 (PE- or FITC-conjugated), anti-CD8 (PE- or APC-conjugated), anti-Mac-1-FITC, anti-CD3-PE, anti-CD25-APC, anti-CD44-PE, anti-Gr-1-PE, and anti-CD19-APC (all from PharMingen). FACS analysis was performed on freshly stained samples.

Isolation and Characterization of Endothelial Cells (ECs).

Livers of Mx-Cre⁺ or Mx-Cre⁻ mice injected with pI-pC 3 weeks prior were used for the isolation of liver ECs. Liver tissues were homogenized and digested in collagenase D and lysates were run through a cell strainer. ECs were enriched by taking the interphase of a 30% Histodenz (Sigma) and RPMI suspension of cells overlaid after spinning at 1500×g for 20 min. Cells were further affinity isolated by CD31-M450 prebound magnetic beads. Isolation of lung ECs and Matrigel morphogenesis assays were carried out as previously described (Balconi et al., 2000) and cells were grown on dishes coated with bovine fibronectin (Sigma). VEGF and basic FGF were obtained from the Biological Resources Branch, NCI Preclinical Repository. Cells were grown in DMEM+0.5% BSA supplemented with 100 ng/ml VEGF, 100 ng/ml FGF, or 10% FBS. EC proliferation and apoptosis were measured with the BrdU labelling and detection kit (Roche) and the In situ cell death detection kit (Roche), respectively per the manufacturer's instruction. Viability of cells after various treatments was measured by MTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assays in 96 well plates. Knockdown of mouse Sprouty2 was performed by transfection using FuGENE6 (Roche) of shRNA constructs provided by Dr. William Hahn, Dana Farber Cancer Institute. The shRNA constructs shSpry2_1 and _2 correspond to clone ID#s TRCN0000007521 and 7523, respectively (The RNAi Consortium/BROAD institute, commercially available from Sigma-Aldrich). Vector (pLKO) alone and pLKO-GFP were used as controls to exclude non-specific responses. Cells were selected for 96 hrs with puromycin (1 μg/ml) and used for further assays.

Microarray Analysis.

Freshly isolated lung ECs and liver ECs were grown on fibronectin-coated plates for 72 hours. RNA was isolated using Trizol (Invitrogen) and the RNeasy mini kit (Qiagen). Gene expression profiling was performed utilizing the Affymetrix 430 2.0 chips. dChip (Li and Wong, 2003) was used to normalize arrays and to compute expression indices. The log transformed expression indices x_(ijk) (i-gene, j-condition, k-replicate) were modeled using a hierarchical empirical Bayes model which assumes (i) x_(ijk)|μ_(ij),σ_(i) ²˜N(μ_(ij),σ_(i) ²); (ii) μ_(ij)|μ₀,τ₀ ²∝1; and (iii) σ_(i) ²|ν₀,ω₀ ²˜Inv˜χ²(ν₀,ω₀ ²). To select genes, the posterior mean of σ_(i) ² for each gene was first estimated using a variance shrinkage estimator (Li and Wong, 2001), σ_(i) ² was then set to its estimated {circumflex over (σ)}_(i) ² and fixed. For comparisons between two conditions (e.g., Mx-Cre⁺ Lung vs. Mx-Cre⁻ Lung), t_(i)=(x _(i1)−x _(i2))/({circumflex over (σ)}_(i)√{square root over (1/K₁+1/K₂)}) (K_(j)—the number of arrays within condition j) were used to rank genes. For comparisons among three or more conditions (e.g., “[Mx-Cre⁺ liver EC>Mx-Cre⁻ liver EC] and [Mx-Cre⁺ Lung >Mx-Cre⁻ Lung]”), random samples μ_(ij) were drawn from N(x _(ij),{circumflex over (σ)}_(i) ²/K_(j)). The empirical frequencies that the pre-specified criterion was satisfied among 1000 Monte Carlo draws were then used to rank genes, and for each gene, one minus the empirical frequency was reported as its score.

FoxO DNA Binding Element Studies.

Differentially expressed genes with RefSeq annotations were analyzed for the presence of FoxO BE. The March 2005 version of mouse genome (NCBI build 34) was used in the analysis. A 3^(rd) order Markov chain was used to model the random background sequence. The FoxO matrix was used to scan the target regions. At each position, its likelihood was compared with the likelihood of the background model. A site was picked as a potential FoxO binding site if the likelihood ratio between the FoxO and background model is greater than 250. Potential FoxO binding sites were filtered further by cross-species conservation using two independent approaches. In the first approach (Method1), a conservation score was computed for each position in the genome. FoxO sites whose mean conservation score was among the top 10% of the genome-wide scores were preserved as the conserved FoxO binding sites. To compute the conservation score, multiple alignments between mouse and 9 vertebrate genomes were downloaded from UCSC genome browser (http://genomes.ucsc.edu). A 50 bp sliding window was used to scan the alignment. For each window, matched base pairs were counted between species i and j (denoted by I_(ij), i=mouse; j=human, rat, dog, cow, or zebrafish), and the total number of columns in the pairwise alignment (denoted by N_(ij)). Percent identities ({circumflex over (θ)}_(ij)=I_(ij)/N_(ij)) were computed, and corresponding z-scores (z_(ij)=({circumflex over (θ)}−{circumflex over (θ)}_(ij))/√{square root over (θ_(ij)(1−θ_(ij))/N_(ij))}) were derived, where θ_(ij) was the percent identity between species i and j in a 1 Mbp surrounding window. The z-scores from five pairwise comparisons (mouse-human, mouse-dog, mouse-cow mouse-chicken, and mouse-zebrafish) were then averaged and converted into the interval [0, 255] to serve as the final conservation score. The higher the score, the more conserved a position was. For each gene, the total number of conserved FoxO sites was shown in Tables 1, 2, 3, and 4, and Log₁₀ (likelihood ratio between the FoxO matrix model and background Markov model) of all conserved FoxO sites were added up and the sum shown.

In the second approach (Method2), ortholog genes from human, dog, cow, chicken, and zebrafish were selected for each mouse gene in the gene list. MLAGAN (Brudno et al., 2003) was used to construct the cross-species alignment of target regions. The FoxO matrix was used to scan each species to get potential FoxO binding sites. If a position in the alignment was identified as FoxO binding site in mouse, human and at least one additional species, it was identified as a 3-species conserved FoxO site.

Quantitative Real-Time PCR Analysis.

RNA was harvested using Trizol (Invitrogen) and the RNeasy kit (Qiagen). RNA was treated with RQ1 RNase-freeDNase (Promega), and cDNA was prepared utilizing Superscript II Rnase H-Reverse transcriptase (Invitrogen). Quantitative real-time PCR was performed on cDNA samples utilizing the Quantitect SYBR Green PCR kit (Qiagen) and was run on the Stratagene Mx3000P. Primer sequences are available upon request.

Western Blot Analysis.

Tissues and cells were lysed with RIM buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% Sodium Deoxycholate, 1 mM EDTA, 0.1% SDS) containing complete mini protease inhibitors (Roche). Western blots were performed utilizing 50 μg of lysate protein, incubated with the following antibodies: anti-FKHR(FoxO1)/AFX(FoxO4) (Cell Signaling Technology) and anti-FKHRL1(FoxO3) (Upstate Biotechnology).

Example 2

Generation and Characterization of Single and Compound FoxO Mutant Mice Reveals Modest Neoplastic Phenotypes.

Each conditional allele retained wild type function as evidenced by normal FoxO expression and lack of a phenotype in homozygous mice and derivative cells (FIGS. 6 and 7, and (Castrillon et al., 2003), see below; data not shown). Germline Cre-mediated recombination produced null alleles for each of the FoxO genes as confirmed by PCR, Southern, and Northern blot analyses (FIGS. 6 and 7). Consistent with previous reports, FoxO1 nullizygosity resulted in embryonic lethality circa E10.5, while FoxO1^(−/+) mice were healthy and fertile (Furuyama et al., 2004; Hosaka et al., 2004). FoxO3^(−/−) mice were viable and outwardly normal but developed mild hemolytic anemia, modest glucose intolerance, and premature female sterility due to global activation of primordial follicles soon after birth, as previously reported (Castrillon et al., 2003; Hosaka et al., 2004). Mice deficient for FoxO4 were healthy and fertile with normal body weight and glucose tolerance (data not shown).

Each allele was backcrossed 3 times onto the FVBn background, after which serial intercrosses generated various experimental cohorts comprised of single and compound germline mutant mice (FoxO3^(−/−); FoxO4^(−/−) and FoxO1^(−/+); FoxO3^(−/−); FoxO4^(−/−)), triple conditional FoxO1^(L/L); FOXO3^(L/L); FOXO4^(L/L) mice with and without the interferon-inducible Mx-Cre transgene (Kuhn et al., 1995), and wild type littermate controls. Overall survival as well as spontaneous and carcinogen-induced tumor formation was monitored in the various cohorts.

In the germline mutant series, aging FoxO1^(+/+) and FoxO1^(−/+) mice (n=31 and 52, respectively) showed no differences in overall survival, tumor incidence and multiplicity (FoxO1^(+/+)=0.9±0.1 versus FoxO1^(−/+)=0.9±0.1 tumors per mouse), and tumor spectrum (primarily lung adenocarcinomas which are common neoplasms in aged FVBn animals) (Table 5 and FIG. 8a ). Similarly, aging FoxO4^(+/+) and FoxO4^(−/−) mice (n=26 and 37, respectively) failed to exhibit significant differences in overall survival, tumor incidence and multiplicity (FoxO4^(+/+), 0.7±0.1; FoxO4^(−/−), 1.0±0.2 tumors per mouse), and tumor spectrum (FIG. 8b , and Table 5). While aged FVBn females are reported to develop occasional prolactinomas (Mahler et al., 1996; Wakefield et al., 2003), 6/19 FoxO4^(−/−) females, generally over 2 years of age, developed this neoplasm compared with 0/11 age-matched FoxO4^(+/+) (p=0.0613) and 2/15 FoxO4^(−/+) (p=0.2569) females. Since FoxOs have been linked to p27^(Kip1) regulation (Medema et al., 2000), it should be emphasized that these prolactinomas are distinct from the intermediate lobe pituitary adenomas arising in p27^(Kip1) and Rb mutant mice (Fero et al., 1996; Hu et al., 1994; Kiyokawa et al., 1996; Mahler et al., 1996; Nakayama et al., 1996; Wakefield et al., 2003). Consistent with a minor tumor suppressor role for FoxO4, treatment with the carcinogen 7,12-dimethylbenz-alpha-anthracene (DMBA) did not reveal significant differences in median survival (23.4 weeks for FoxO4^(−/−) and 27.3 weeks for FoxO4^(+/+), p=0.4926) or in tumor incidence or spectrum, although FoxO4^(−/−) animals showed a modest increase in lymphoma incidence that approached statistical significance (p=0.053, Fisher Exact Test) (FIGS. 10a and b ).

Aged FoxO3^(−/−) mice are prone to dermatitis between 10-24 months of age and 15/35 FoxO3^(−/−) mice required euthanasia due to progressive skin ulceration (FIGS. 8c and d ). The basis of this dermatitis has not been explored. Of the remaining dermatitis-free FoxO3^(−/−) mice, 19/20 mice developed tumors (FIGS. 9c and d , p=0.0010; FIGS. 8e and f ). While FoxO3^(−/−) females show a more rapid onset of tumors, this relates to increased occurrence of ovarian stromal neoplasms (4/16 FoxO3^(−/−) versus 0/10 FoxO3^(+/+) females; p=0.1358) and a shorter latency and increased growth of pituitary prolactinomas (7/16 FoxO3^(−/−) versus 1/10 FoxO3^(+/+) females; p=0.0989) (FIGS. 9e and f , Table 5) which are likely due to elevated gonadotropins associated with premature ovarian failure (Castrillon et al., 2003) and the predisposition of aged FVB/n females to prolactinomas (Mahler et al., 1996; Wakefield et al., 2003). Finally, FoxO3 status did not alter the outcome of DMBA exposure studies with respect to overall survival and tumor incidence and spectrum (FIGS. 10 c and d), with the exception of angiosarcomas, which had an increased incidence in FoxO3−/+(p=0.036) and −/− (p=0.364) versus +/+ animals. Thus, FoxO3 deficiency is associated with a modest tumor phenotype that emerges very late in life and is dominated by neoplasms driven by an abnormal hormonal milieu in females, and a modest increase in the incidence of angiosarcomas but not overall cancer incidence following carcinogen treatment.

To begin to address possible redundancy among FoxO family members, compound germline mutant mice and controls were generated and subjected to detailed long-term phenotypic analyses. FoxO3^(−/−) FoxO4^(−/−) mice (n=31), followed for up to 100 weeks of age, did not exhibit new phenotypes, change in overall survival, or exacerbation of physiological anomalies beyond those observed in the FoxO3^(−/−) mice (e.g., hemolytic anemia, ovarian failure, etc), or increased tumor incidence (FIG. 11). Lastly, through 80 weeks of observation, FoxO1^(−/+) FoxO3^(−/−) FoxO4^(−/−) mice do not display changes in tumor incidence and spectrum (data not shown). These studies included exhaustive comparisons with FoxO3^(+/+) FoxO4^(+/+) and FoxO3^(−/+) FoxO4^(−/−) controls (As noted above, the detailed tumor incidence, spectrum, and overall survival data pertaining to these cohorts are presented in FIGS. 8-11, and Table 5).

Vascular Neoplasms in Aging Mx-Cre⁺ Mice of Various FoxO Genotypes

Discrete liver hemangiomas and occasional angiosarcomas were seen in aged Mx-Cre⁺; FoxO1^(L/L) (1 mouse (at 85 weeks) of 5 mice that died at 53-100 weeks and 3 of 4 mice examined at 87-104 weeks of age), Mx-Cre⁺;FoxO1/3^(L/L) (7 of 10 mice that died at 50-82 weeks and 2 of 2 mice examined at 90-102 weeks) and Mx-Cre⁺;FoxO1/4^(L/L) mice (5 of 7 mice that died at 58-86 weeks and 2 of 2 mice examined at 96-98 weeks).

Methods

Targeting constructs. FoxO3^(−/−) mice were generated as described (Castrillon et al., 2003). The FoxO1 and FoxO4 genomic regions encompassing both coding exons were cloned and mapped from a bacterial artificial chromosome library. The targeting vector pKOII (courtesy Nabeel Bardeesy) carries a negative selection marker for diphtheria toxin (DT), a positive selection marker for neomycin resistance (Neo), Frt sites (white rectangles) and loxP sites (gray triangles). For FoxO4, the first coding exon (containing the start codon and encoding the N-terminal half of the full-length protein) was targeted. For FoxO1, the second major coding exon (encoding the C-terminal half of the full-length protein) was targeted. TC1 embryonic stem cells were electroporated and selected transfected cells by standard techniques. 96 clones were screened for each of the three loci using a genomic probe external to the targeting construct (FIGS. 6 and 7) to identify recombinants that contained the Neo selection cassette and both loxP sites. Blastocyst injections were carried out and transmitting chimeric mice were bred with EIIa-Cre transgenic (Lakso et al., 1996) mice to generate the null alleles, and with ACT-FlpE mice (courtesy of Susan Dymecki) to generate the conditional/floxed alleles. Each allele was backcrossed three times to FVB/n females and progeny of these matings that were Cre⁻ and Flp⁻ were then intercrossed with littermates to generate the experimental cohorts. Mice were genotyped by multiplex PCR (primers and conditions are available on request).

Generation of Mice for Aging and Tumorigenesis Studies.

Due to the location of FoxO4 on the X chromosome, it is not possible to generate FoxO4^(−/+) males or littermate FoxO4^(+/+) and −/− females. Otherwise, all experimental and control animals were matched littermates. For the Mx-Cre studies, an Mx-Cre⁺ male (courtesy of Klaus Rajewsky) was mated with a FoxO1^(L/L); FoxO3^(L/L); FoxO4^(L/L) (FoxO1/3/4^(L/L)) female, and the progeny were then crossed with FoxO1/3/4^(L/L) mice. The resulting offspring were intercrossed to generate mice of the desired genotypes. Litters were treated starting at 4-5 weeks with 3 intraperitoneal injections every other day of 300 μg of polyinosine-polycytidylic acid (pI-pC), a synthetic analog of double-stranded RNA (Invivogen), to induce expression of interferon-beta, thereby leading to transient activation of Mx-Cre.

Whole Animal and Tumor Analysis.

Littermate controls were analyzed for tumor-free survival. Animals were genotyped by PCR and allowed to age undisturbed in a maximum of 5 mice per cage with standard chow and water ad libitum in a standard light-day cycle. Mice were monitored three times a week and were sacrificed when moribund or if external tumors exceeded 2 cm in diameter and scored as a death in survival analysis. Those animals with cancer, as determined by histological analysis, were scored as an event in the tumor-free survival Kaplan-Meier analysis. For lung tumors, analysis of tumor load was carried out as done previously (Zhang et al., 2001).

DMBA Treatment.

Seven-day old pups were treated with 5% 7,12-dimethylbenz-alpha-anthracene (DMBA) in acetone by pipetting 50 μl onto the back, and allowing it to air-dry. Mice were monitored three times a week for signs of morbidity and underwent full autopsies for tumor detection.

Analysis of Rosa26-lacZ Reporter Mice.

B6, 129-GtRosa26^(tm1Sor) mice (no. 3309, Jackson Laboratory, Bar Harbor, Me.) were used as a reporter strain. Organs were harvested from an Mx-Cre⁺; Rosa26-lacZ⁺ female and an Mx-Cre⁻; Rosa26-lacZ⁺ female (both injected with pI-pC) and cut into 1-2 mm pieces. After fixation with 4% paraformaldehyde+0.25% glutaraldehyde in PBS for 2 hours, tissues were stained for 24 hours in the dark and postfixed in formalin. Tissues were embedded in paraffin and sections were stained with Nuclear Fast Red or left unstained.

Chromatin Immunoprecipitation (ChIP) Assay.

Two million liver ECs were crosslinked by addition of 1% formaldehyde to the medium for 10 min followed by quenching with 125 mM glycine. The cells were resuspended in lysis buffer (1% SDS, 10 mM EDTA, and 50 mM Tris (pH 8.1), Protease Inhibitor Cocktail II (Roche)), sonicated 10 times for 30 s with 2 min idle time, the lysates were cleared by centrifugation. One hundred microliters of the sheared DNA was diluted 10-fold in dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl (pH 8.1), and 167 mM NaCl). Chromatin solution was precleared for 1 h at 4° C. with 60 μl of protein G-agarose/salmon sperm DNA. Ten microliters of the precleared chromatin solutions was saved for assessment of input chromatin, and the rest of the precleared chromatin solutions was incubated with 1 ug of anti-RNA polymerase II (clone CTD4H8, Upstate) or mixture of anti-FoxO Ab (Afx, FKHR, Cell signalling, FKHRL1, Upstate) overnight at 4° C. Immune complexes were collected on 60 μl of protein A/G Plus-agarose/salmon sperm beads. Precipitates were washed sequentially for 5 min each in Low salt wash buffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 150 mM NaCl], High salt wash buffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 500 mM NaCl], LiCl immune wash buffer (Upstate). Precipitates were then washed twice with 1×TE (pH. 8.0) and extracted two times with 1% SDS, 0.1 M NaHCO₃. Elutes were incubated at 65° C. with 0.25 M NaCl overnight to reverse cross-linking followed by another 1 hr incubation at 45° C. with 10 μM EDTA, 40 μM Tris-HCl (pH 6.8) and 2 μg Proteinase K (Sigma). The DNA was purified using a PCR purification kit (Qiagen) with 40 μl of distilled water. Two microliters of immunoprecipitated DNA was used for real time PCR analysis in 25-μl total reaction volumes, and the following primers were used in the ChIP assays:

Amplicon forward (5′-3′), reverse (5′-3′) a CATTTGTGTGTTTTGAGGGAGAGAT, CGGCAGTTGGGTTGGAATTA b TAGGGCGACTCAGTGGCTATC, GACCGGAGTCAAAGGACCTTC c AATTAGCAAATGGCTCCCGG, TTTGTGACTGTGCCATGAAGC d TTCCAGTCCTCCAAGCAATCTAG, AGTGCCTCCAGGAAGGGAAT PCR conditions were as follows: 30 cycles of 94° C. for 30 s, 55° C. for 30 s, and 72° C. for 30 s. Twenty microliters of PCR products were analyzed by 2% TBE agarose gel containing ethidium bromide.

Table 5 shows the tumor spectra of mice with germline mutation of a single FoxO gene. For pituitary adenomas and female-specific tumors, mice are separated by gender for analysis of incidence.

Table 6 shows a list of the top 600 differentially expressed genes (˜300 up or down) from Mx-Cre⁺ and Mx-Cre⁻ thymocytes.

Table 7 shows a list of the top 600 genes (˜300 up or down) showing greater fold changes in liver EC from Mx-Cre⁺ and Mx-Cre⁻ compared to no significant changes in lung EC

TABLE 1 FoxO targets in thymocyte # of Conservation # of 3-species Gene name BE(s) Score BE(s) CIS Upregulated FoxO Target Genes Sox5 25 71 6 Bt127 Rora 14 39 4 Dacna2d1 13 37 3 Creb5 11 31 3 Igsf4d 11 32 3 Srgap3 10 29 3 Pfkfb2 7 19 3 Pacs1 6 17 3 Sox4 5 14 3 Evil6 Mmp16 11 33 2 Slc12a1 9 25 2 Dcamkl1 6 17 2 4931406l20Rik 6 17 2 4931406120Rik Scn5a 5 15 2 Sourl1 4 12 2 Pou4f3 4 11 2 Shox2 11 32 1 Hoxd12 6 19 1 Id4 6 17 1 Hapln1 6 17 1 Slc12a1 5 14 1 Golph4 5 14 1 Slc24a2 5 15 1 Ppp1r9a 5 15 1 Barx2 5 15 1 Ddx47 4 11 1 Abcd2 4 12 1 Npy2r 3 9 1 Shrm 3 8 1 Igfbp3 3 8 1 Flt1 2 6 1 Dyx1c1 2 5 1 D14Ertd171e 2 5 1

TABLE 2 FoxO targets in thymocyte # of Conservation # of 3-species Gene name BE(s) Score BE(s) CIS Downregulated FoxO Target Genes Npas3 10 29 6 Cdh8 10 31 4 Myef2 6 16 3 Cdkn1b 5 14 3 Dlgap1 10 29 2 Bt114 Tmem25 8 23 2 Lrrtm3 7 20 2 Tbx4 5 14 2 Kcnj2 5 16 2 AJ430384 4 12 2 Efna5 4 12 2 Rest 8 22 1 Odz3 7 19 1 Dpf3 5 15 1 Bag5 5 13 1 Cacna1e 4 11 1 Olfm3 3 9 1 Gas7 3 9 1 Tmem71 3 9 1 Znrf4 3 8 1 Aqp7 2 6 1 C730036D15Rik 2 6 1 Kcnip4 2 6 1 Usp3 2 6 1 Evi98 Myo1e 2 6 1 Tgfbr2 2 6 1 Cdh23 2 6 1 Trem2 2 6 1 Klc3 1 3 1 Cryab 1 3 1 Cacna2d2 1 3 1 Ncbp2 1 3 1 Dmrt3 1 3 1

TABLE 3 FoxO targets in Liver EC # of Conservation # of 3-species Gene name BE(s) Score BE(s) CIS Upregulated FoxO Target Genes Meist 10 28 4 Evi6 Pbx1 11 32 3 Tsc22d1 3 9 2 Ccnd1 8 25 1 Ccndl Sdpr 4 11 1 Sepm 4 12 1 Fbn1 4 12 t 8mper 4 12 1 Rab34 3 9 1 D0H4S114 3 9 1 Pcolce 2 6 1 Id1 1 3 1

TABLE 4 FoxO targets in Liver EC # of Conservation # of 3-species Gene name BE(s) Score BE(s) CIS Downregutated FoxO Target Genes Spry2 10 28 5 Tcf4 10 27 4 Klf6 10 28 3 Cited2 6 17 2 Adm 5 15 2 Ccrn4l 4 12 1 Hmga2 1 3 1 Mcc1 1 3 1 Ctgf 1 3 1 

1-99. (canceled)
 100. A method of assessing a) whether a subject is afflicted with thymic lymphoma or a hamartomatous tumor, the method comprising comparing: i) the level of expression of at least one or more markers in a subject sample, wherein said one or more markers is selected from any one of Tables 1-4; and ii) the normal level of expression of said one or more markers in a control non-cancer sample, wherein a significant modulation in the level of expression of said one or more markers in the subject sample and the normal level is an indication that the subject is afflicted with thymic lymphoma or the hamartomatous tumor; b) the progression of thymic lymphoma or a hamartomatous tumor in a subject, the method comprising: i) detecting in a subject sample at a first point in time the expression of one or more markers; ii) repeating step i) at a subsequent point in time; and iii) comparing the level of expression of said one or more markers detected in steps i) and ii), and therefrom monitoring the progression of thymic lymphoma or the hamartomatous tumor; wherein at least one or more of said markers is selected from the group consisting of the markers listed in Tables 1-4; c) the efficacy of a test compound for inhibiting thymic lymphoma or a hamartomatous tumor in a subject, the method comprising comparing: i) expression of one or more thymic lymphoma or hamartomatous tumor markers in a first sample obtained from the subject and exposed to the test compound; and ii) expression of said one or more markers in a second sample obtained from the subject, wherein the second sample is not exposed to the test compound, wherein at least one or more of said markers is selected from the group consisting of the markers listed in Tables 1-4, and a significantly modulated level of expression of one or more of said markers in the first sample, relative to the second sample, is an indication that the test compound is efficacious for inhibiting thymic lymphoma or the hamartomatous tumor in the subject; d) the efficacy of a therapy for inhibiting thymic lymphoma or a hamartomatous tumor in a subject, the method comprising comparing: i) expression of one or more thymic lymphoma or hamartomatous tumor markers in a first sample obtained from the subject prior to providing at least a portion of therapy to the subject, and ii) expression of said one or more markers in a second sample obtained from the subject following provision of the portion of the therapy, wherein at least one or more of said markers is selected from the group consisting of the markers listed in Tables 1-4, and a significantly modulated level of expression of at least one or more of said markers in the second sample, relative to the first sample, is an indication that the therapy is efficacious for inhibiting thymic lymphoma or the hamartomatous tumor in the subject; e) a composition for inhibiting thymic lymphoma or a hamartomatous tumor in a subject, the method comprising: i) obtaining a sample comprising cancer cells from the subject; ii) separately exposing aliquots of the sample in the presence of a plurality of test compositions; iii) comparing expression of one or more thymic lymphoma or hamartomatous tumor markers in each of the aliquots; and iv) selecting one of the test compositions which alters the level of expression of one or more of the markers in the aliquot containing the test composition, relative to other test compositions; wherein at least one or more of said markers is selected from the group consisting of the markers listed in Tables 1-4; f) the potential of a test compound to give rise to thymic lymphoma or a hamartomatous tumor, the method comprising: i) maintaining separate aliquots of cells derived from any tissue in the presence and absence of the test compound; and ii) comparing expression of one or more thymic lymphoma or hamartomatous tumor markers in each of the aliquots, wherein at least one or more of said markers is selected from the group consisting of the markers listed in Tables 1-4, and a significantly modulated level of expression of said one or more markers in the aliquot maintained in the presence of the test compound, relative to the aliquot maintained in the absence of the test compound, is an indication that the test compound possesses potential to give rise to thymic lymphoma or the hamartomatous tumor; g) whether thymic lymphoma or a hamartomatous tumor has metastasized in a subject, the method comprising comparing: i) the level of expression of one or more thymic lymphoma or hamartomatous tumor markers in a subject sample, and ii) the normal level or non-metastatic level of expression of said one or more markers in a control sample wherein at least one or more of said markers is selected from the group consisting of the markers listed in Tables 1-4, and a significant difference between the level of expression of said one or more markers in the subject sample and the normal level or non-metastatic level is an indication that the thymic lymphoma or the hamartomatous tumor has mestastasized; h) the aggressiveness or indolence of thymic lymphoma or a hamartomatous tumor comprising comparing: i) the level of expression of one or more thymic lymphoma or hamartomatous tumor markers in a sample, and ii) the normal level of expression of said one or more markers in a control sample, wherein at least one or more of said markers is selected from the group consisting of the markers listed in Tables 1-4, and a significant difference between the level of expression of said one or more markers in the sample and the normal level is an indication that the thymic lymphoma or the hamartomatous tumor is aggressive or indolent; i) a composition capable of modulating thymic lymphoma or a hamartomatous tumor, the method comprising i) obtaining a sample comprising cancer cells; ii) contacting said cells with a test compound; and iii) determining the ability of the test compound to modulate the amount and/or activity of one or more markers, wherein the one or more markers is selected from the group consisting of the markers listed in Tables 1-4, thereby identifying a modulator of thymic lymphoma or the hamartomatous tumor; or j) a composition capable of modulating thymic lymphoma or a hamartomatous tumor, the method comprising i) contacting one or more markers selected from the group consisting of the markers listed in Tables 1-4 with a test compound; and ii) determining the ability of the test compound to modulate the amount and/or activity of a marker selected from the group consisting of the markers listed in Tables 1-4, thereby identifying a composition capable of modulating thymic lymphoma or the hamartomatous tumor.
 101. A method of inhibiting a) thymic lymphoma or a hamartomatous tumor in a subject, the method comprising: i) obtaining a sample comprising cancer cells from the subject; ii) separately maintaining aliquots of the sample in the presence of a plurality of test compositions; iii) comparing expression of one or more thymic lymphoma or hamartomatous tumor markers in each of the aliquots; and iv) administering to the subject at least one of the test compositions which alters the level of expression of said one or more markers in the aliquot containing that test composition, relative to other test compositions, wherein at least one or more of said markers is selected from the group consisting of the markers listed in Tables 1-4; b) thymic lymphoma or a hamartomatous tumor in a subject at risk for developing thymic lymphoma or a hamartomatous tumor, the method comprising enhancing or inhibiting expression of one or markers selected from the group consisting of the markers listed in Tables 1-4; or c) thymic lymphoma or with a hamartomatous tumor in a subject, the method comprising providing to cells of the subject an antisense oligonucleotide complementary to a polynucleotide encoded by one or markers selected from the group consisting of the markers listed in Tables 1-4, or a segment of said polynucleotide.
 102. A kit for assessing a) whether a subject is afflicted with thymic lymphoma or a hamartomatous tumor, the kit comprising reagents for assessing expression of one or more thymic lymphoma or hamartomatous tumors markers, wherein at least one or more of said markers is selected from the group consisting of the markers listed in Tables 1-4; b) the presence of thymic lymphoma or hamartomatous tumor cells, the kit comprising a nucleic acid probe which specifically binds with a transcribed polynucleotide encoded by one or markers selected from the group consisting of the markers listed in Tables 1-4; c) the suitability of each of a plurality of compounds for inhibiting thymic lymphoma or a hamartomatous tumor in a subject, the kit comprising: i) the plurality of compounds; and ii) a reagent for assessing expression of one or more thymic lymphoma or hamartomatous tumor markers, wherein at least one or more of said markers is selected from the group consisting of the markers listed in Tables 1-4; d) the presence of human thymic lymphoma or hamartomatous tumor cells, the kit comprising an antibody, wherein the antibody specifically binds with a protein encoded by one or more markers selected from the group consisting of the markers listed in Tables 1-4, or a fragment of said protein; or e) the potential of a test compound to give rise to thymic lymphoma or a hamartomatous tumor, the kit comprising cells derived from any tissue and a reagent for assessing expression of one or more markers selected from the group consisting of the markers listed in Tables 1-4. 